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Steady state kinetic analysis of the transcription of cloned Drosophila melanogaster tRNA[sup Ser] genes St. Louis, Daniel Claude 1984

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STEADY STATE KINETIC ANALYSIS OF THE TRANSCRIPTION OF Cap CLONED DROSOPHILA MELANOGASTER tRNA GENES by Daniel Claude St. Louis B.Sc, M c G i l l University, 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Faculty of Graduate Studies Department of Biochemistry University of B r i t i s h Columbia accept t h i s thesis as conforming to the required standard The University of B r i t i s h Columbia © Daniel Claude St. Louis August, 1984 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of ^ i ' a c l a ^ i j / - ^  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date dMr /s~ jjfsY E - 6 (3/81) (ii) Abstract Drosophila melanogaster Schneider II c e l l s contain a f a c t o r which i n h i b i t s i n v i t r o t r a n s c r i p t i o n of cloned tRNA genes in crude extracts made from these c e l l s . The i n h i b i t o r could, however, be e f f e c t i v e l y neutralized by addition of c e r t a i n nontemplate DNAs. In the absence of the t r a n s c r i p t i o n i n h i b i t o r a c t i v i t y , the steady state k i n e t i c s of tRNA production from cloned genes followed one-substrate enzyme k i n e t i c s to a high degree of accuracy. I n i t i a l and maximal rates of t r a n s c r i p t i o n and apparent a f f i n i t y constants were analyzed for a c o l l e c t i o n of cloned D. melanogaster tRNA genes. The s t a b i l i t y of the complex formed by the t r a n s c r i p t i o n proteins and the template DNA was found to be nearly constant for the genes examined. The t r a n s c r i p t i o n rates, however, were greatly influenced by the DNA sequences flanking the tRNA genes. Analysis of t r a n s c r i p t i o n competition between DNA templates showed pure competitive behavior. I n h i b i t i o n constants derived from these experiments indicated that the formation of the t r a n s c r i p t i o n complex was affected by sequences flanking the tRNA genes. Furthermore, the rate l i m i t i n g step in complex formation was independent of the s t a b i l i t y of the f i n a l form of the complex. (iii) Table of Contents Page Abstract (ii) Table of Contents (iii) L i s t of Tables (vii) L i s t of Figures (viii) Acknowledgements (xili) Abbreviations (xil) Introduction 1 I. The Transcription Apparatus 1 I I . Structure of Eukaryotic RNA Polymerases 2 I I I . Eukaryotic Genes 3 A. Class I genes 3 B. Class II genes 4 C. Class I II genes 5 1. RNA polymerase I I I t r a n s c r i p t i o n systems 6 2. Tr a n s c r i p t i o n control region f o r Class I I I genes . . . 8 a) 5S RNA genes 8 b) tRNA genes 9 3. Ki n e t i c s 13 a) Competition between 5S RNA genes 14 b) Competition between tRNA genes 16 c) Conclusions 19 (iiii) Page 4. Formation of t r a n s c r i p t i o n complexes 19 a) Implications in the regulation of 5S RNA 22 genes iii vivo IV. Present Investigations 24 Materials and Methods 27 I. General 27 A. I s o l a t i o n of recombinant plasmid DNA . . 27 B. Digestion of DNA with r e s t r i c t i o n endonucleases 28 C. End l a b e l l i n g of DNA 29 D. Agarose gel electrophoresis 29 E. Polyacrylamide gel electrophoresis 29 F. Autoradiography . . . • ^ ^.-^ 30 G. I s o l a t i o n of nucleic acid from polyacrylamide gels: E l e c t r o e l e c t i o n 31 H. I s o l a t i o n of nucleic acid from agarose gels: Trough e l u t i o n 31 32 I. Synthesis of [5*- P]pCp 32 J. 3' end l a b e l l i n g of tRNA . 32 K. Southern trans f e r of DNA 33 L. Hybridization of [5'- 3 2P]pCp l a b e l l e d tRNA 33 M. Plasmid DNA 33 N. Treatment of 3' extended ends with T DNA polymerase . . 33 0. Preparation of l i n e a r , blunt ended pUC8 34 < v) Page P. L i g a t i o n of DNA r e s t r i c t i o n fragments to l i n e a r i z e d pUC8 34 Q. Transformation of E. c o l i (JM83) 35 R. Selection of cloned fragments 35 S. Rapid small scale i s o l a t i o n of plasmid DNA 36 T. P u r i f i c a t i o n of plasmid DNA for DNA sequence analysis . . . 38 U. DNA sequence analysis 38 V. In v i t r o t r a n s c r i p t i o n and analysis of RNA products . . . . 40 Results 41 I. Construction of recombinant plasmids 41 A. pUC8 as a cloning vector 41 B. Subcloning of two tRNA genes contained in pDtl6 . . . 44 C. Construction of pDt73x27 73 D. Construction of pDtBH27 86 I I . Properties of Drosophila (Schneider II) c e l l - f r e e extracts . . 96 A. T r a n s c r i p t i o n products derived from tRNA genes . . . . 96 B. Authenticity of RNA t r a n s c r i p t s directed by pDt5 101 C. E f f e c t of DNA concentration on the rate of t r a n s c r i p t i o n 104 Ser D. Kinetic analysis of tRNA genes 112 E. T r a n s c r i p t i o n k i n e t i c s of plasmids containing more than one gene 137 (vO Page S6£* F. Tra n s c r i p t i o n k i n e t i c s of plasmids between tRNA Val genes and a tRNA gene 146 G. Kin e t i c s of t r a n s c r i p t i o n : Time course assay 168 H. Kinetics of t r a n s c r i p t i o n : Enzyme concentration curve . . 172 I. Kinetics of t r a n s c r i p t i o n : Extract dependence 172 Discussion 180 Ser I. Transcription of Drosophila tRNA genes 180 II . Drosophila Schneider II c e l l free-extracts contain an i n h i b i t o r . 182 I I I . Kinetics of t r a n s c r i p t i o n i n the presence of nontemplated DNA 184 IV. Kinetic analysis of competing templates 191 V. Model for t r a n s c r i p t i o n of tRNA genes 194 References 202 (vii) L i s t of Tables Page Table I. In s i t u h y b r i d i z a t i o n of plasmid DNAs containing Ser tRNA genes 25 Table I I . Summary of the size of the tRNA gene t r a n s c r i p t i o n products 99 Table I I I . Kinetic parameters from i n v i t r o t r a n s c r i p t i o n Ser of the tRNA genes 135 Table IV. Kin e t i c parameters from i n v i t r o t r a n s c r i p t i o n of S G r plasmids containing more than one tRNA gene . . . 144 Table V. Comparison of the k i n e t i c parameters from the in v i t r o Seir t r a n s c r i p t i o n of tRNA genes derived from pDtl6 . . 145 Table VI. Comparison of K i , Kmapp and Vo 165 Table VII. Ki n e t i c parameters measured i n d i f f e r e n t extracts . . . . 178 (vi ii) L i s t of Figures Page Figure 1. P a r t i a l r e s t r i c t i o n endonuclease map of the sequenced Sec* portion of pDtl6 containing two tRNA genes 42 Figure 2. Determination of the A l u l and Hhal r e s t r i c t i o n endonuclease fragments which contain a tRNA gene . . 45 Figure 3. Analysis of the pDtl6 subclones by r e s t r i c t i o n endonuclease cleavage 50 Se JC Figure 4. Possible orientations of the tRNA genes on the 510 bp A l u l DNA fragments 53 Figure 5. DNA sequence analysis of pDtA16-2#l: A. Forward Direct i o n 57 Figure 6. DNA sequence analysis of pDtA16-2#l: B. Reverse D i r e c t i o n 59 Figure 7. DNA sequence analysis of pDtA16#3: A. Forward D i r e c t i o n 61 Figure 8. DNA sequence analysis of pDtA16#3: B. Reverse Direct i o n 63 Figure 9. DNA sequence analysis of pDtH16#l: A. Forward Direct i o n 65 Figure 10. DNA sequence analysis of pDtH16#l: B. Reverse Direct i o n 67 Figure 11. DNA sequence analysis of pDtH16#5: A. Forward Direct i o n 69 Figure 12. DNA sequence analysis of pDtH16#5: B. Reverse Direct i o n 71 ( v i i i i ) Page Ser Figure 13. The strategy used to reconstruct a tRNA^^ gene containing a termination sequence 74 Figure 14. I d e n t i f i c a t i o n of DNA fragments derived from pDt27 Set* and pDt73 which contain tRNA genes 77 Figure 15. I d e n t i f i c a t i o n of pDt73x27 hybrid plasmids 82 Figure 16. Determination of pDt73x27 hybrid plasmid organization . . 84 Figure 17. DNA sequence analysis of pDt73x27 #44 and #54 87 Figure 18. I d e n t i f i c a t i o n of a Hindlll-BamHI fragment from Ser pDt27 containing tRNA^^, genes 91 Figure 19. DNA sequence analysis of pDtBH27#l 94 Figure 20. Transcription of d i f f e r e n t plasmids carrying Se 2? Drosophila tRNA genes 97 Sec Figure 21. Authenticity of the tRNA gene t r a n s c r i p t i o n products 102 Figure 22. E f f e c t of DNA concentration on the rate of t r a n s c r i p t i o n of pDt5 and pDtH16#5 105 Figure 23. Tr a n s c r i p t i o n of pDt5 in the presence of nontemplate DNA 109 Figure 24. Tr a n s c r i p t i o n of pDt5 in the presence and absence of pBR322 113 Figure 25. Tr a n s c r i p t i o n of pDtl7R in the presence and absence of pBR322 116 Figure 26. Tr a n s c r i p t i o n of pDtA16-2#l in the presence and absence of pBR322 119 ( X) Page Figure 27. Tra n s c r i p t i o n of pDtH16#5 in the presence and absence of pBR322 122 Figure 28. Tra n s c r i p t i o n of pDtA16-2#3 in the presence and absence of pBR322 125 Figure 29. Tra n s c r i p t i o n of pDtH16#l in the presence and absence of pBR322 128 Figure 30. Tra n s c r i p t i o n of pDt73x27 in the presence and absence of pBR322 131 Figure 31. Tr a n s c r i p t i o n of pDtl6 in the presence and absence of pBR322 138 Figure 32. Tra n s c r i p t i o n of pDt27 in the presence and absence of pBR322 140 Figure 33. Tra n s c r i p t i o n of..pDtBH27#l in the presence and absence of pBR322 142 Figure 34. Tra n s c r i p t i o n competition between a tRNA^^^ Val gene and a tRNA gene 147 Val Figure 35. Tra n s c r i p t i o n competition between a tRNA Ser , c n gene and a tRNA ? 7 7 gene 150 Figure 36. Tra n s c r i p t i o n competition between a tRNA^^ Val gene and a tRNA gene 153 Se jt* Figure 37. Tra n s c r i p t i o n competition between a tRNA^^ Va l gene without a terminator sequence and a tRNA ^ gene i 156 Figure 38. E f f e c t of pDt5 DNA on the t r a n s c r i p t i o n of pDt0.3 DNA 159 (xi> Page Figure 39. E f f e c t of pDt0.3 DNA on the t r a n s c r i p t i o n of pDt5 DNA 161 Figure 40. E f f e c t of pDt0.3 DNA on the t r a n s c r i p t i o n of pDt73x27 DNA 163 Figure 41. Time course assay 169 Figure 42. Extract concentration curve 173 Figure 43. Extract dependence on t r a n s c r i p t i o n 175 Figure 44. Model for t r a n s c r i p t i o n of tRNA genes 179 (xii) Abbreviations Used bp - base pairs DNA - deoxyribonucleic acid RNA - ribonucleic acid DNase - deoxyribonuclease RNase - ribonuclease Sexr tRNA - serine transfer r i b o n u c l e i c acid Val tRNA - valine t r a n s f e r r i b o n u c l e i c acid M - molecular weight r EDTA - ethylenediamine t e t r a a c e t i c acid (xiii) Acknowledgements I t i s a pleasure to thank George B. Spiegelman, Gordon Tener and Robert C. M i l l e r , J r . f o r t h e i r guidance, advice, c r i t i c i s m , support, encouragement and many stimulating discussions. In addition, I would l i k e to acknowledge the members of the Spiegelman laboratory f o r t h e i r patience and support, e s p e c i a l l y L. Duncan f o r preparation of the S - 1 0 0 extracts. I would also l i k e to thank Linda f o r enduring. Last, but not l e a s t , I would l i k e to thank my mother and father, f o r without t h e i r help I would not be here. 1 Introduction I. The Transcription Apparatus In prokaryotes, a single species of RNA polymerase catalyzes a l l RNA synthesis. The enzyme binds to s p e c i f i c s i t e s on the DNA template c a l l e d promoters found 5' to a l l b a c t e r i a l t r a n s c r i p t i o n units (1). A f t e r promoter recognition, the b a c t e r i a l RNA polymerase assumes an active conformation and i n i t i a t e s RNA synthesis. The RNA i s elongated by the polymerization of ribonucleoside triphosphates to the 3' OH of the growing chain. Elongation continues u n t i l the t r a n s c r i p t i o n complex reaches a termination s i g n a l which tr i g g e r s the d i s s o c i a t i o n of RNA polymerase from the DNA template and the RNA chain i s released (2). Transcription in eukaryotes i s s i m i l a r to that in prokaryotes however, the o v e r a l l process i s considerably more complex. The eukaryotic c e l l possesses 3 chromatographically d i s t i n c t RNA polymerases designated RNA polymerase I, II and I I I . Each enzyme transcribes a d i f f e r e n t class of gene (3, 4, 5, 20, 21). The DNA-dependent RNA polymerases are active in two locations within the c e l l nucleus, the nucleolus and the nucleoplasm. In the nucleolus, RNA polymerase I i s responsible for the synthesis of 18S RNA, 28S RNA and 5.8S RNA and accounts f o r 50 - 70% of the t o t a l nuclear a c t i v i t y . In the nucleoplasm c l o s e l y associated with chromatin, RNA polymerase II c a r r i e s out the synthesis of heterogenous nuclear RNA (the precursor to messenger RNA) representing 20 - 40% of the t o t a l t r a n s c r i p t i o n . RNA polymerase I I I , also found in the nucleoplasm, accounts for the smallest proportion of the c e l l u l a r t r a n s c r i p t i o n a l a c t i v i t y (10 - 20%). Genes which are transcribed by RNA polymerase III 2 include 5S RNA (77, 78), tRNA (69, 99, 100, 109) adenovirus v i r u s -associated RNA (VA RNA) (63, 112) and human Alu family genes (123). I I . Structure of Eukaryotic RNA Polymerases The multiple forms of RNA polymerase have been i s o l a t e d and characterized from a v a r i e t y of eukaryotic organisms (reviewed in 22). The three s t r u c t u r a l l y d i s t i n c t polymerases are of high molecular weight (M R - 470,000 - 700,000) and consist of 10 - 15 subunits. Even though the classes d i f f e r markedly in structure, there i s immunological evidence to suggest that they may have r e l a t e d subunits (23, 24). When chromatographically p u r i f i e d , the enzymes do not e x h i b i t t h e i r c h a r a c t e r i s t i c functions as expected from in vivo analysis. Polypeptides which do not co-purify with the enzymes are necessary to d i r e c t accurate and s e l e c t i v e t r a n s c r i p t i o n of the s p e c i f i c classes of genes (6 - 9, 22, 47, 48, 86). In addition to the d i s t i n c t s t r u c t u r a l and f u n c t i o n a l properties, the eukaryotic RNA polymerases can be distinguished by t h e i r d i f f e r e n t i a l s e n s i t i v i t y to the fungal toxin, a-amanitin; d i r e c t i n t e r a c t i o n of the toxin with RNA polymerase (19) blocks the elongation step of the t r a n s c r i p t i o n cycle (25). RNA polymerase of one class i s o l a t e d from d i f f e r e n t organisms have s i m i l a r structure, d i f f e r i n g by only 1 or 2 polypeptides. These enzymes share s i m i l a r general properties; i o n i c strength optima, divalent metal ion requirement, template preference and s e n s i t i v i t y to a-amanitin (22). 3 I I I . Eukaryotic Genes In the c e l l each class of enzyme recognizes template structure and nucleotide sequences common to the genes transcribed by that polymerase. Since p u r i f i e d eukaryotic RNA polymerases do not s e l e c t i v e l y and accurately transcribe p u r i f i e d template, analysis of a p u r i f i e d t r a n s c r i p t i o n system would reveal l i t t l e relevant information about the mechanisms of promoter recognition and t r a n s c r i p t i o n i n i t i a t i o n and termination. To mimic the in vivo s i t u a t i o n , ni v i t r o t r a n s c r i p t i o n systems using c e l l - f r e e extracts from a v a r i e t y of sources have been developed (see below). Such systems were used to elucidate DNA sequences and protein factors required for gene expression. A. Class I genes The products from Class I genes, 18S, 5.8S and 28S ribosomal RNA are important s t r u c t u r a l components of the ribosome and account f o r the majority of the t o t a l RNA synthesized in the c e l l . To accomplish the massive synthesis of ribosomal RNA, the eukaryotic genome contains about 200 copies of each gene organized i n cl u s t e r s of tandem repeats which are transcribed by RNA polymerase I in the nucleolus. Some eukaryotes expand t h e i r protein synthetic machinery by increasing the copy number of the ribosomal RNA genes (30 - 33). Amplification of the ribosomal RNA genes in these organisms generates large amounts of extrachromosomal ribosomal DNA which i s t i g h t l y associated with the nucleolus. T r a n s c r i p t i o n of each repeating unit by the nucleolar RNA polymerase y i e l d s a single large precursor RNA molecule, 40 - 45S in s i z e . This large t r a n s c r i p t i s 4 processed to generate the 3 ribosomal RNAs (28, 29). I n i t i a t i o n of t r a n s c r i p t i o n occurs 450 - 650 bp upstream from the 18S ribosomal RNA coding region. Recently, several investigators have determined the nucleotide sequences required for s p e c i f i c i n i t i a t i o n of t r a n s c r i p t i o n by RNA polymerase I f o r mouse (10), Xenopus (11) and Drosophila (12) ribosomal RNA genes. These sequences surround the s i t e s of i n i t i a t i o n of t r a n s c r i p t i o n and include the regulatory elements but because they lack sequence homology no consensus sequence can be drawn. The k i n e t i c s of ribosomal RNA synthesis i s reminiscent of the k i n e t i c s of RNA polymerase III t r a n s c r i p t i o n systems ( k i n e t i c s of RNA polymerase III t r a n s c r i p t i o n are discussed below; 10). However, unlike RNA polymerase I I I t r a n s c r i p t i o n systems, d e t a i l s about the protein factors involved i n the formation of t r a n s c r i p t i o n complexes on Class I genes are not known. P u r i f i c a t i o n of these factors should provide more information about the molecular mechanisms of the i n i t i a t i o n process. B. Class II genes The organization of promoter sequences encoded by Class II genes appears to be very much l i k e b a c t e r i a l promoters (34) in that at l e a s t 2 control elements precede the gene. A conserved sequence re f e r r e d to as the Goldberg-Hogness or "TATA" box i s located 25 to 30 nucleotides upstream from the i n i t i a t i o n s i t e of Class II genes. This sequence appears to a f f e c t the rate of RNA synthesis as well as the s i t e of i n i t i a t i o n (35 - 38). A second control region located approximately 50 nucleotides further upstream influences only the rate of t r a n s c r i p t i o n 5 (39 - 41). Enhancer elements positioned upstream from the two c o n t r o l regions also a f f e c t s the rate of t r a n s c r i p t i o n (15). Since the rate and e f f i c i e n c y of t r a n s c r i p t i o n varies among Class II promoters (Class II genes) the r e l a t i v e contribution of the two control regions on the processes of t r a n s c r i p t i o n may depend on the DNA sequence and structure surrounding these regions. The regulation of many Class II genes observed in vivo cannot be reproduced in c e l l - f r e e extracts suggesting that the factors or mechanisms required for regulation are not functioning in c e l l - f r e e systems. Perhaps the regulatory signals are mediated through a combination of the following: 1) the presence of p o s i t i v e or negative e f f e c t o r molecules, 2) DNA modification, or 3) chromatin structure (13 - 15). C. Class III genes Transcription of Class III genes i s the best characterized of the 3 classes of eukaryotic genes. Early studies showed that a p u r i f i e d reconstituted t r a n s c r i p t i o n system containing p u r i f i e d RNA polymerase I I I i s o l a t e d from Xenopus l a e v i s or mammalian tissues and cloned 5S RNA genes or adenovirus v i r u s associated RNA genes cannot support accurate or s e l e c t i v e t r a n s c r i p t i o n even though RNA polymerase III does maintain polymerizing a c t i v i t y (42 - 46). However, oocyte chromatin templates d i r e c t 5S RNA synthesis when transcribed with p u r i f i e d RNA polymerase I I I . F a i t h f u l t r a n s c r i p t i o n of adenovirus virus associated RNA genes on nuclear adenovirus templates i s achieved by addition of exogenous mammalian enzyme (63, 112). These studies demonstrate that s p e c i f i c 6 t r a n s c r i p t i o n of Class I I I genes i n a reconstituted system requires one or both of the following: 1) addit i o n a l factors which appear to be stably associated with chromatin, or nuclear templates, and 2) some modification of the template DNA which occurs i n vivo. Since p u r i f i e d 5S RNA genes (51, 52) and tRNA genes (53 - 57) microinjected into Xenopus oocytes d i r e c t s p e c i f i c synthesis of 5S RNA and tRNA, the DNA template contains the necessary information to support accurate and s e l e c t i v e t r a n s c r i p t i o n . Recent f r a c t i o n a t i o n and re c o n s t i t u t i o n experiments have i d e n t i f i e d several factors in addition to RNA polymerase I I I which are es s e n t i a l f o r the t r a n s c r i p t i o n of Class I I I genes (to be discussed below) (47 - 50). 1. RNA polymerase I I I t r a n s c r i p t i o n systems With the development of c e l l - f r e e extracts that mediate the t r a n s c r i p t i o n of exogenous, p u r i f i e d genes, the analysis of Class I I I gene expression i n v i t r o has provided valuable insight into the mechanisms of t r a n s c r i p t i o n , i n p a r t i c u l a r , information about the DNA sequences and the protein factors involved in these processes. I n i t i a l l y , investigations by Wu demonstrated that a post-mitochondrial f r a c t i o n (20,000 x g) from human KB c e l l s accurately transcribed p u r i f i e d adenovirus template (58). Subsequent investigations by other laboratories resulted in the development of two d i f f e r e n t t r a n s c r i p t i o n systems that d i r e c t e d accurate and s e l e c t i v e t r a n s c r i p t i o n of cloned 5S RNA genes: 1) an extract made from germinal v e s i c l e from Xenopus oocyte nuclei (59) and 2) a supernatant f r a c t i o n r e s u l t i n g from a high speed spin (100,000 x g) of whole Xenopus 7 oocytes (S-100) (62). At present, S-100 extracts prepared from many sources (47, 60 - 65) are used to f a i t h f u l l y transcribe 5S RNA (60, 62, 64, 65), tRNA (61, 64 - 66), VA RNA (58, 60, 63, 64) and Alu family genes (67, 68). Hela c e l l extracts (63) and Drosophila Kc c e l l extracts (11) have been shown to contain a t r a n s c r i p t i o n i n h i b i t o r . Addition of vector pBR322 to t r a n s c r i p t i o n reactions containing template DNA remove the e f f e c t of the i n h i b i t o r and stimulate the rate of t r a n s c r i p t i o n . The t r a n s c r i p t i o n of a v a r i e t y of Class III genes from d i f f e r e n t organisms in homologous or heterologous c e l l - f r e e systems demonstrates a lack of species s p e c i f i c i t y in the in v i t r o expression of these genes. The permissiveness of heterologous t r a n s c r i p t i o n reactions indicates a strong evolutionary conservation in the o v e r a l l t r a n s c r i p t i o n apparatus; that i s , conservation of factors as well as DNA sequences required for e f f i c i e n t t r a n s c r i p t i o n . Like Class II genes, t r a n s c r i p t i o n of Class I I I genes i s not regulated in homologous c e l l - f r e e systems. To date the best documented example of the lack of gene s p e c i f i c regulation in homologous c e l l - f r e e extracts i s the e f f i c i e n t t r a n s c r i p t i o n of both oocyte and somatic 5S RNA genes in somatic extracts derived from d i f f e r e n t Xenopus tis s u e s . In vivo expression of oocyte 5S RNA genes in somatic tissues i s r e s t r i c t e d . Recent studies suggest that sequences 5* to tRNA genes may be important for organism s p e c i f i c expression (61, 69, 70). 8 2. Transcription control region f o r Class III genes Because the c l a s s i c a l d e f i n i t i o n of a promoter i s an extragenic c i s acting regulatory sequence (71), the i n i t i a l approach employed to i d e n t i f y f u n c t i o n a l l y important DNA sequences which mediate RNA polymerase I I I t r a n s c r i p t i o n was to search f or sequence homologies at the 5' or 3' borders of several Class I II genes (72, 94). A comparison of the DNA sequences preceding the genes revealed l i t t l e or no sequence homology suggesting that these sequences do not play a major r o l e in promoter recognition. In contrast, sequences following Class I II genes are r e l a t i v e l y conserved. This analysis implies that e i t h e r intragenic sequences or 3' flanking sequences are d i r e c t l y involved in the processes of t r a n s c r i p t i o n . Subsequent investigations (discussed below) have shown that intragenic sequences and not flanking sequences are necessary for accurate and s e l e c t i v e t r a n s c r i p t i o n . Employing recombinant DNA technology to generate a series of 5' and 3' deletion mutants of the o r i g i n a l DNA template allows the i d e n t i f i c a t i o n of regions in the DNA sequences which a f f e c t gene function. The r e s u l t s of such experiments (described below) demonstrate that the sequences encoded by Class I I I genes required f o r RNA polymerase I I I recognition are unique and quite d i f f e r e n t from conventional b a c t e r i a l promoters, a) 5S RNA genes To define the control region which d i r e c t s the s p e c i f i c i n i t i a t i o n of 5S RNA synthesis, Bogenhagen et a l . (77) and Sakonju et a l . (78) generated a series of deletions of a fragment of Xenopus borealis somatic 5S DNA from ei t h e r the 5* or the 3* side of the gene. When transcribed, 5' end 9 deletions extending as f a r as 50 nucleotides into the gene continue to support d i s c r e t e 5S RNA sized t r a n s c r i p t s ; however, deletions 55 or more nucleotides into the gene no longer support 5S RNA synthesis. These res u l t s suggest that deletions of 55 or more nucleotides destroy an e s s e n t i a l region located between the nucleotides 50 and 55 which i s required to mediate s p e c i f i c i n i t i a t i o n of t r a n s c r i p t i o n by RNA polymerase I I I . The removal of the 5* flanking sequence does not eliminate the a b i l i t y of the 5S RNA gene to d i r e c t t r a n s c r i p t i o n i n i t i a t i o n , but deleting these sequences to less than 26 nucleotides upstream from the gene dramatically reduces the rate of t r a n s c r i p t i o n (79). Similar analysis using a series of 3' deletion mutants define the 3' border of the intragenic control region between the nucleotides 80 and 83. In addition to defining the 3' boundary of the control element, signals encoded in the DNA sequence which are involved in the termination of t r a n s c r i p t i o n are also i d e n t i f i e d . Deletion of the c l u s t e r of 4 T residues located 3' to the gene a f f e c t termination of t r a n s c r i p t i o n without a f f e c t i n g i n i t i a t i o n . Thus demonstrating the importance of t h i s T c l u s t e r (52, 80). A T to C t r a n s i t i o n mutation in one of the T residues also affected termination of t r a n s c r i p t i o n . The presence of a s i m i l a r c l u s t e r of T residues in other Class I I I genes has been shown to be necessary for termination of t r a n s c r i p t i o n (73 - 75). b) tRNA genes The approach employed for defining the control region in 5S RNA genes has also been used to analyze the DNA sequences surrounding and i n t e r n a l to eukaryotic tRNA genes which a f f e c t the processes of t r a n s c r i p t i o n . The 10 f i r s t l i n e of evidence which suggested that tRNA genes, l i k e 5S RNA genes, contained an intragenic control element came from the following investigations. When injected into Xenopus oocytes or when used as template in oocyte extracts, DNA fragments containing tRNA genes from Xenopus (72), Bombyx (61, 95) and Drosophila (96) in which the 5' flanking sequence had been deleted to within 15 bp, 6 bp or 0 bp of the s t r u c t u r a l gene res p e c t i v e l y , s t i l l supported t r a n s c r i p t i o n . When DNA fragments Met containing the 5' and the 3' end of a Xenopus tRNA gene were tested for t h e i r a b i l i t y to be transcribed, Kressman et a l . (97) found that neither the 5* portion nor the 3' portion of the tRNA gene could d i r e c t tRNA synthesis. In addition, n a t u r a l l y occurring point mutations found to Tyr a f f e c t the expression of a yeast Sup4 tRNA gene i_n vivo (98) and in  v i t r o (75) mapped within the s t r u c t u r a l gene. The most deleterious of a l l the mutations examined i s a single base pair change at p o s i t i o n 56 of the suppressor tRNA gene which completely abolishes the a b i l i t y of the gene to promote t r a n s c r i p t i o n . The control regions within tRNA genes are mapped by systematically deleting the 5' and 3' DNA sequences (69, 99 - 101) in experiments analogous to those by Bogenhagen et^ a l . (77, 78) on 5S RNA genes. The deletion analysis of these tRNA genes reveal that unlike the intragenic control element in 5S RNA genes, the promoter in tRNA genes i s s p l i t into two sequence blocks which act in concert to y i e l d accurate and e f f i c i e n t t r a n s c r i p t i o n . These sequence blocks designated the D-control region and the T-control region consist of the nucleotides 8-25 (the D-stem and D-loop of the mature tRNA) and 50 - 58 (the T-stem and T-loop) 11 r e s p e c t i v e l y , by the standard numbering system for tRNA genes (101) and correspond to highly conserved sequences occuring in most eukaryotic tRNAs (102). The high degree of homology found in the D- and T-control regions between tRNA genes may r e f l e c t the conservation of the nucleotides important for promoting t r a n s c r i p t i o n as well as the structure and function of the tRNA i t s e l f . Since tRNA genes containing v a r i a b l e loops or n a t u r a l l y occurring intervening sequences (104) are e f f i c i e n t l y transcribed, the DNA region between the two control elements can be varied in both length or sequence. Hofstetter et a l . (69) emphasized the f l e x i b i l i t y of the tRNA t r a n s c r i p t i o n mechanism by showing that a l t e r a t i o n s in the length of the Met DNA region between the s p l i t promoter of a Xenopus tRNA gene or replacement of the region with synthetic DNA l i n k e r s d i d not dramatically a f f e c t t r a n s c r i p t i o n . However, C i l i b e r t o et a l . (103) observed that only Pro moderate a l t e r a t i o n s of the spacer region in the centre of a tRNA gene from C. elegans were tolerated without severe reduction i n t r a n s c r i p t i o n . These apparently c o n f l i c t i n g r e s u l t s suggests that perhaps the a b i l i t y of a tRNA gene to t o l e r a t e sequence v a r i a b i l i t y between the D-and T-control region and maintain t r a n s c r i p t i o n e f f i c i e n c y depends on promoter strength. Although sequences e s s e n t i a l for the expression of tRNA genes are i n t e r n a l , the t r a n s c r i p t i o n a l a c t i v i t y and the s i t e of i n i t i a t i o n of a given tRNA gene i s dependent on the nature of the DNA sequences upstream from that gene. Deletions and substitutions of 5' flanking sequences of tRNA genes markedly e f f e c t both the rate of t r a n s c r i p t i o n and the s i t e of 12 i n i t i a t i o n (57, 105). The wild type DNA sequence 5' to a Bombyx tRNA gene i s absolutely required for the expression of th i s gene in a homologous extract (61, 70). The loc a t i o n of the 5' c o n t r o l l i n g element e s s e n t i a l f o r t r a n s c r i p t i o n (between 34 and 11 nucleotides before the s i t e of t r a n s c r i p t i o n i n i t i a t i o n ) suggests a requirement of t h i s region to d i r e c t l y i n t e r a c t with the RNA polymerase I II during i n i t i a t i o n . In contrast, a G-T r i c h DNA sequence found to precede several Drosophila LiVS tRNA genes i s i n h i b i t o r y when positioned 13 nucleotides i n front of the s t r u c t u r a l gene (105). Deletion or repositioning of the i n h i b i t o r y sequence leads to r e l i e f of t r a n s c r i p t i o n i n h i b i t i o n iri v i t r o . A 9 base pair G-C r i c h a l t e r n a t i n g purine-pyrimidine t r a c t i s also found to i n h i b i t Met t r a n s c r i p t i o n . Maximum i n h i b i t i o n of a variant Xenopus tRNA gene i s observed when the i n h i b i t o r y sequence i s located 11 bp 5* to the s t r u c t u r a l gene (106, 107). The i n h i b i t o r y e f f e c t exerted by t h i s sequence i s removed upon de l e t i o n . Stretches of al t e r n a t i n g purine-pyrimidine sequence as short as 8 and 10 base pairs have been implicated in the formation of Z DNA in supercoiled SV40 and plasmid DNA (108). The presence of an al t e r n a t i n g purine-pyrimidine t r a c t at or near the s i t e of i n i t i a t i o n of tRNA synthesis may a l t e r the DNA conformation of the DNA sequence such that i t can no longer i n t e r a c t favorably with the RNA polymerase I I I t r a n s c r i p t i o n complex. The e f f e c t of the 5' flanking sequence on the r e l a t i v e rates of tRNA synthesis from a v a r i e t y of tRNA genes may r e f l e c t a l t e r n a t i v e sets of signals recognized by the t r a n s c r i p t i o n complex in d i f f e r e n t organisms. A 13 comparison of the a c t i v i t i e s of tRNA genes in heterologous and homolgous extracts supports t h i s idea (69, 101, 109). 3. Kinetics Due to the complexity of the c e l l free system used to support t r a n s c r i p t i o n of Class III genes, knowledge about the molecular mechanisms of t r a n s c r i p t i o n i s l i m i t e d to the k i n e t i c analysis of various template DNAs and information regarding the p a r t i a l l y p u r i f i e d factors required by the t r a n s c r i p t i o n apparatus (reviewed below). Kinetic analysis of genes transcribed by RNA polymerase I I I i s dependent on both the type of gene transcribed and the source of the c e l l - f r e e extract used. Extracts prepared from Xenopus nuclei (54) or whole oocytes (62, 47) display biphasic t r a n s c r i p t i o n k i n e t i c s when 5S RNA genes are used as template. The time course for t r a n s c r i p t i o n i n these extracts show a pronounced lag in 5S RNA synthesis followed by a period of rapid RNA synthesis. However, when tRNA genes are used as template (95) or when 5S RNA genes are allowed to preincubate with extract p r i o r to t r a n s c r i p t i o n , no lag in RNA synthesis i s observed (59). Human c e l l - f r e e extracts supporting the synthesis of 5S RNA or adenovirus VA RNA displays no lag period (60). In contrast, extracts made for both Drosophila Kc c e l l s and Hela c e l l s e x h i b i t biphasic k i n e t i c s i n which the length of the lag period i s shown to be temperature dependent (110, 111). Presumably, in extracts which support rapid RNA synthesis without a lag phase, the factors required f o r i n i t i a t i o n of t r a n s c r i p t i o n are in excess or the processes involved in complex formation are very rapid. The presence of a 14 lag in product formation may indicate that modification of the DNA template i s required for t r a n s c r i p t i o n (59, 62) or a l t e r n a t i v e l y may r e f l e c t the time required to form gene-stable t r a n s c r i p t i o n complexes. Some of these extracts may contain stimulating factors which enhance complex assembly. Analyzing the a b i l i t y of de l e t i o n mutants to d i r e c t accurate and s e l e c t i v e t r a n s c r i p t i o n i i i v i t r o defines the borders of the intragenic control elements of Class I I I genes. Q u a l i t a t i v e measurements provided by these analyses detect the presence or absence of accurate t r a n s c r i p t i o n i n i t i a t i o n events without determining q u a n t i t a t i v e l y the changes in the t r a n s c r i p t i o n system caused by deletion mutations. A d e t a i l e d comparative study in which the rate of t r a n s c r i p t i o n of each mutant along with the a b i l i t y of these mutants to compete with "wild type" genes f o r components necessary for RNA synthesis provides a d d i t i o n a l information about the events leading to s p e c i f i c t r a n s c r i p t i o n . This experimental approach measures q u a n t i t a t i v e l y the contribution of the intragenic control elements and surrounding sequences to the o v e r a l l t r a n s c r i p t i o n e f f i c i e n c y . This approach has been applied to analyze mutant adenovirus VA RNA genes (63, 112), mutant 5S RNA genes (79), and mutant tRNA genes from Xenopus (97), Bombyx (61) and more recently Drosophila (110, 111). a) Competition between 5S RNA genes By measuring the a b i l i t y of 5' and 3' deletion mutants of Xenopus 5S RNA genes to compete with "wild type" 5S RNA genes for t r a n s c r i p t i o n factors found in homologous c e l l - f r e e extracts, Wormington et a l . (79) 15 were able to i d e n t i f y the nucleotide sequences which influence the competitive strength of these genes. Competition experiments using a series of 3* d e l e t i o n mutants revealed that sequences downstream from residue 97 (upstream from the normal termination s i t e f o r 5S RNA genes) have no influence on the competitive strength, the s i t e of i n i t i a t i o n (77) nor the binding of the t r a n s c r i p t i o n factor TFIIIA (see below, 113). However, deletions from either side of the gene extending well into the control element a f f e c t both the competitive strength of the template and the rate of RNA synthesis. In addition, the competitive strength of somatic 5S RNA genes but not oocyte 5S RNA genes are s e n s i t i v e to 5' flanking sequence deletions suggesting that sequences at or around the s i t e of i n i t i a t i o n of somatic 5S RNA genes determine the e f f i c i e n c y and accuracy of RNA synthesis. Although intact oocyte and somatic 5S RNA genes transcribe at the same rate, the two genes compete with each other with d i f f e r e n t e f f i c i e n c y i n d i c a t i n g that the rate of t r a n s c r i p t i o n i s not necessarily r e l a t e d to competitive strength (see also 114). The reduced competitive strength of oocyte 5S DNA templates r e l a t i v e to somatic templates i_n v i t r o appears to be due to s p e c i f i c base pair differences between i t and the somatic gene. The synthesis of 5S RNA genes in oocyte nuclear extracts i s l i m i t e d due to the low l e v e l s of an e s s e n t i a l t r a n s c r i p t i o n f a c t o r TFIIIA (see below). Addition of p u r i f i e d TFIIIA to the extracts stimulates s p e c i f i c t r a n s c r i p t i o n of 5S RNA genes but not other Class III genes (87). Supplementing the extract with p u r i f i e d RNA polymerase has no a f f e c t (115). The a v a i l a b i l i t y of RNA polymerase III and possibly other 16 components which are common to the t r a n s c r i p t i o n of 5S RNA genes, Xenopus tRNA genes and adenovirus VA RNA genes may explain the reason why these genes compete weakly with each other i n th i s extract. b) Competition between tRNA genes To indicate the extent of sequences within tRNA genes that are required f o r t r a n s c r i p t i o n e f f i c i e n c y and competitive strength and the re l a t i o n s h i p of these sequences to the two intragenic control regions, S o i l and his co-workers analyzed a series of 5' and 3' deletion mutants of At* Or a Drosophila tRNA gene for t h e i r a b i l i t y to support t r a n s c r i p t i o n and i n h i b i t synthesis of a reference tRNA gene (10,111,116). They found that an int a c t gene consisting of the two control regions (the D-control region and the T-control region representing the e s s e n t i a l sequences) along with 5' and 3' flanking sequences was required for both optimum t r a n s c r i p t i o n and maximum competitive strength. Systematically deleting the 5* flanking sequence of the tRNA gene seemed to have a more profound e f f e c t on the rate of t r a n s c r i p t i o n than on the competitive strength of the mutant genes. When the 5' flanking sequence was deleted to within 10 nucleotides from the coding region the rate of t r a n s c r i p t i o n of t h i s mutant gene was markedly reduced while i t s competitive strength was apparently unaffected. When such deletions extended into the D-control region of the tRNA A r g gene, leaving only the T-control region and 3* flanking sequences i n t a c t , mutant genes were s t i l l able to compete with the reference gene a l b e i t at a lower l e v e l . Reciprocal experiments i n which the 3' flanking sequences were systematically removed resulted in 17 mutant genes which progressively l o s t t h e i r a b i l i t y to compete in t r a n s c r i p t i o n reactions. The competitive strength of the tRNA gene was completely abolished when 3' deletions extended into the T-control region. This i n a b i l i t y of DNA templates without the T-control region to compete in t r a n s c r i p t i o n reactions suggests the t r a n s c r i p t i o n a l requirement of a f a c t o r , analogous to TFIIIA, which binds to the T-control region of the tRNA gene. Possible evidence of a second f a c t o r perhaps binding to th D-control region i s obtained from studies which show that A.CR Leu the 5' halves of a Drosophila tRNA gene and a S. c e r e v i s i a e tRNA gene (117) s t i l l support low l e v e l RNA synthesis, but are unable to compete. ArK To determine the e f f e c t s of tRNA gene deletions on the formation of tRNA gene-stable t r a n s c r i p t i o n complexes (111,116), a modified form of the competition assay i s employed. Since Drosophila Kc c e l l - f r e e extract exhibit a lag phase of approximately 30 min., the events leading to the rapid synthesis of tRNA are rather slow. As a method of following the events occurring during the lag phase which lead to product formation, competitor templates are preincubated in t r a n s c r i p t i o n extracts for various times p r i o r to addition of the reference gene. Using t h i s assay, Schaak et a l . ( I l l ) showed that a stable t r a n s c r i p t i o n complex forms on Ars the tRNA gene within 5 min. of preincubation which does not d i s s o c i a t e during 2 hours of t r a n s c r i p t i o n . The formation of the stable complex i s s u f f i c i e n t to d i r e c t template usage but not s u f f i c i e n t to promote rapid RNA synthesis. Monitoring the a b i l i t y of d e l e t i o n mutants Ar& of the tRNA gene to exclude t r a n s c r i p t i o n of the i n t a c t gene reveals 18 that formation of a stable t r a n s c r i p t i o n complex i s absolutely dependent on the T-control region while sequences 3' to the gene (inc l u d i n g the t r a n s c r i p t i o n termination s i t e ) , the D-control region and 5' flanking sequences contribute s i g n i f i c a n t l y to the s t a b i l i t y . A d e t a i l e d analysis of the 5' delet i o n mutants suggest that modulatory sequences e x i s t in the 5' flanking sequence (as far upstream as 60 nucleotides from the s t r u c t u r a l gene) which a f f e c t either the a f f i n i t y of the template for the t r a n s c r i p t i o n apparatus or the rate of t r a n s c r i p t i o n , or both. The properties exhibited by Hela c e l l extracts t r a n s c r i b i n g tRNA A r (* genes with respect to 5' and 3* flanking sequences as well as intragenic requirements i n stable complex formation and t r a n s c r i p t i o n e f f i c i e n c y are si m i l a r but not i d e n t i c a l to Drosophila Kc c e l l extracts (116). Systematic removal of the 5' flanking sequence r e s u l t s in the reduction of the t r a n s c r i p t i o n e f f i c i e n c y without correspondingly a f f e c t i n g the competitive strength in both c e l l - f r e e t r a n s c r i p t i o n systems. When these deletions extend to within 10 bp of the s t r u c t u r a l gene, Drosophila Kc c e l l extracts no longer support t r a n s c r i p t i o n whereas Hela c e l l extracts d i r e c t t r a n s c r i p t i o n at 50% reduced e f f i c i e n c y . The competitive strength of t h i s template i n both t r a n s c r i p t i o n systems i s only s l i g h t l y affected. Deletions entering the 5' end of the tRNA gene r e s u l t i n a progressive reduction in the a b i l i t y of these DNAs to compete in the two extracts. Although these del e t i o n mutants are not transcribed in the Drosophila system, Hela c e l l extracts continue to support t r a n s c r i p t i o n of mutants i n which deletions extend 7 bp into the coding sequence. Hela c e l l extracts and Drosophila c e l l extracts show s i m i l a r dependence on 3' flanking 19 sequences. When 3' flanking sequences are deleted, the a b i l i t y to compete and to transcribe i s reduced. Once deletions enter the T-control region these a b i l i t i e s are completely l o s t . c) Conclusions In summary, k i n e t i c analysis of Class I I I genes provided valuable information about the DNA sequences e s s e n t i a l in promoting RNA synthesis, modulating t r a n s c r i p t i o n e f f i c i e n c y and a f f e c t i n g t r a n s c r i p t i o n complex s t a b i l i t y . These studies also reveal evidence for a putative T-control factor and possibly a D-control f a c t o r which by binding to t h e i r respective control regions in tRNA genes would allow promoter recognitions by RNA polymerase I I I and other t r a n s c r i p t i o n f a c t o r s . A d e t a i l e d DNA sequence analysis of 5S RNA genes, adenovirus VAI RNA genes and tRNA genes has revealed that although these genes are s t r u c t u r a l l y unique there are i n t r i n s i c blocks of sequence homology which may r e f l e c t t r a n s c r i p t i o n f a c t o r ( s ) requirements for promoter recognition shared by these genes. The tRNA genes (118, 119) and VAI RNA genes (63, 120) share sequence homology within the two noncontiguous sequence blocks representing the control regions. A sequence homologous to the D-control region of tRNA genes i s contained within the 34 nucleotide intragenic c o n t r o l region of 5S RNA genes (6, 119). 4. Formation of t r a n s c r i p t i o n complexes Details about the actual mechanisms and proteins involved in complex formation or t r a n s c r i p t i o n i n i t i a t i o n requires the p u r i f i c a t i o n and 20 c h a r a c t e r i z a t i o n of the components of the t r a n s c r i p t i o n apparatus. In an attempt to uncover some of the underlying mechanisms involved in the processes of t r a n s c r i p t i o n Roeder and his colleagues have frac t i o n a t e d Xenopus and human c e l l - f r e e extracts by chromatographic methods (47, 48, 50, 91). Early investigations demonstrated that accurate and s e l e c t i v e t r a n s c r i p t i o n of Class III genes by p u r i f i e d RNA polymerase I I I required the addition of other t r a n s c r i p t i o n f a c t o r s . Two p a r t i a l l y p u r i f i e d factors designated, Factor IIIB and Factor IIIC along with RNA polymerase were necessary to i n i t i a t e synthesis of tRNA and adenovirus VA RNA whereas f a i t h f u l t r a n s c r i p t i o n of 5S RNA genes required an a d d i t i o n a l component termed Factor IIIA (TFIIIA). The 5S gene-specific f a c t o r , TFIIIA, has been p u r i f i e d to homogeneity and has been shown to bind to the intragenic control region of 5S RNA genes (47, 87, 91). Perhaps the i n t e r a c t i o n of one or more of these factors with the conserved sequences present in tRNA genes and adenovirus VA RNA genes and with the homologous region found in 5S RNA genes involves promoter recognition. Other f a c t o r s , including RNA polymerase I I I , form the complete t r a n s c r i p t i o n complex a f t e r the i n i t i a l i n t e r a c t i o n . Stable t r a n s c r i p t i o n complexes in crude extracts have been shown to form on p u r i f i e d templates before i n i t i a t i o n of t r a n s c r i p t i o n and have been found to p e r s i s t for many rounds of t r a n s c r i p t i o n (77, 111, 116). The number and the i d e n t i t y of the chromatographically p u r i f i e d factors necessary to form gene-stable t r a n s c r i p t i o n complexes are determined by the a b i l i t y of a template incubated with l i m i t i n g amounts of p u r i f i e d 21 f a c t o r to preclude the t r a n s c r i p t i o n of a second template added subsequently to the reaction (50). Factor IIIC was found to be an i n t e g r a l component f o r gene-stable complex formation on a l l Class I I I genes. Although Factor IIIC bound d i r e c t l y to tRNA and VA RNA genes, the binding of t h i s f a c t o r to 5S RNA genes required Factor IIIA. Alone Factor IIIA formed a metastable complex with 5S RNA genes which was completely s t a b i l i z e d by the addition of Factor IIIC. The two f a c t o r s , IIIA and IIIC bind to the 5S DNA template independently and are stably associated with the template for a single round of t r a n s c r i p t i o n . Both Factor IIIB and Factor IIIC are necessary for stable complex formation on tRNA genes. During t r a n s c r i p t i o n of tRNA genes in these reconstituted systems, Factor IIIB appears to cycle. In the absence of Factor IIIB, Factor IIIC interacts weakly with the tRNA gene and r e a d i l y d i s s o c i a t e s . Despite the t r a n s c r i p t i o n requirement of adenovirus VA RNA genes f o r Factor IIIB, Factor IIIC alone was s u f f i c i e n t to form a gene-stable complex. Met The 3' h a l f of a tRNA gene forms an exclusionary complex with Factor IIIC thereby i n h i b i t i n g VA RNA and 5S RNA synthesis from competing templates. Factor IIIC behaves much l i k e the putative T-control f a c t o r proposed by S o i l and his group (110) which interacts with the T-control region of tRNA genes (50). R e s t r i c t i o n endonuclease protection studies using VA RNA genes as template suggest that Factor IIIC i n t e r a c t s with the intragenic control region within VA RNA genes homologous to the T-control region of tRNA genes. Since a sequence homologous to the T-control region has not yet been observed in 5S RNA genes, the involvement of Factor IIIC in the formation of a stable t r a n s c r i p t i o n complex on 5S RNA genes i s not 22 c l e a r . Perhaps Factor IIIC recognizes the 5S DNA template only a f t e r association of the template with Factor IIIA (50). a) Implications i n the regulation of 5S RNA genes in vivo Class III genes share at le a s t 3 common t r a n s c r i p t i o n f a c t o r s , factors IIIB, IIIC and RNA polymerase I I I . The r e l a t i v e a f f i n i t y of the in d i v i d u a l factors f o r s p e c i f i e d genes may in part determine the expression of a c e r t a i n set of genes. Once established, stable t r a n s c r i p t i o n complexes are maintained for many rounds of t r a n s c r i p t i o n and are r e s i s t a n t to competition (50, 121). The d i f f e r e n t i a l expression of Xenopus 5S RNA genes during development implicates the involvement of gene-specific Factor IIIA and the other common components i n regulation. The 20,000 oocyte 5S RNA genes (81, 82) per haploid genome in Xenopus  la e v i s are present i n a l l c e l l types but are only expressed during oogenesis. Oocytes require massive synthesis of proteins during early development thus 5S RNA, an important component of ribosomes, must also be present in large q u a n t i t i e s . When the conversion process of the oocyte into a f e r t i l i z e d egg i s complete, oocyte 5S RNA synthesis stops. During b l a s t u l a formation, the somatic 5S RNA genes are activated (83 - 85) and account for almost a l l 5S RNA synthesis throughout development. The r a t i o of the rate of oocyte to somatic 5S RNA synthesis changes 1000 f o l d during development (93). Factor IIIA (TFIIIA) i s o l a t e d from Xenopus (86) oocytes has a fo u r f o l d greater a f f i n i t y f o r somatic than oocyte 5S RNA genes (89). I t is u n l i k e l y that TFIIIA acts alone as a developmental regulatory factor 23 despite i t s involvement in the i n i t i a t i o n of the t r a n s c r i p t i o n i n oocytes. However, i t i s i n t r i g u i n g that the l e v e l of TFIIIA and the rate of 5S RNA synthesis are both high in oocytes and f a l l r a p i d l y during maturation (86, 88, 90). This r e l a t i o n s h i p between the rate of 5S RNA synthesis and the l e v e l of TFIIIA may r e f l e c t the requirement for 5S RNA and therefore, TFIIIA, at any p a r t i c u l a r time during development and not necessarily the type of 5S RNA gene transcribed. In addition to binding to 5S RNA genes, TFIIIA also binds to 5S RNA i n v i t r o suggesting that t r a n s c r i p t i o n of 5S RNA genes may be subject to autoregulation (87, 88) and may be r e s t r i c t e d to previously activated genes. While studying the assembly of 5S RNA genes into stable t r a n s c r i p t i o n a l l y active or inactive complexes iii v i t r o . Brown and his colleagues recognized that these complexes resembled the t r a n s c r i p t i o n states of 5S RNA genes in somatic c e l l chromatin (121, 122). When chromatin was i s o l a t e d from somatic c e l l s and transcribed in v i t r o , oocyte SS RNA genes remained inactive while somatic 5S RNA genes supported RNA synthesis. Treatment of the chromatin with high s a l t concentrations resulted in the a c t i v a t i o n of oocyte 5S RNA gene s p e c i f i c t r a n s c r i p t i o n (see also 89, 93). Although the e f f e c t of t h i s s a l t concentration on Xenopus chromatin structure remains to be resolved i t i s possible that such treatment may allow the rearrangement of chromatin-associated molecules r e s u l t i n g in the a c c e s s i b i l i t y of oocyte 5S RNA genes for RNA polymerase I I I t r a n s c r i p t i o n apparatus. Excluding the e f f e c t s of chromatin structure on gene expression, the establishment of gene-stable t r a n s c r i p t i o n complexes may play an important r o l e in the maintenance of a 24 d i f f e r e n t i a t e d state (121). Since the 20,000 oocyte 5S RNA genes are located in large blocks near the telomeres of most i f not a l l the chromosomes in Xenopus l a e v i s (92), chromatin structure surrounding 5S RNA genes may also be important in the regulation of 5S RNA synthesis. In somatic c e l l s , the telomeres appear to be condensed into heterochromatin (92). I t i s possible that early i n oogenesis the telomeres are not associated with heterochromatic structure thus allowing f o r s e l e c t i v e developmental expression of oocyte 5S RNA genes. IV. Present Investigations I have investigated the irt v i t r o t r a n s c r i p t i o n of a group of cloned Ser tRNA genes from Drosophila melanogaster i n i t i a l l y i s o l a t e d and characterized by Dunn et a l . (129). These genes were further characterized by Cribbs (130) and Newton (139). Cribbs also showed that Ser Ser tRNA 7 and tRNA ^ are 95% homologous d i f f e r i n g only i n three Se r of 85 nucleotides; one being in the anticodon. Out of the seven tRNA genes contained on f i v e d i f f e r e n t plasmids, six of the genes were found Ser Ser clustered at a.major s i t e of tRNA^ and tRNA^ , h y b r i d i z a t i o n , 12 DE on the X chromosome (Table I ) . The seventh gene coding f o r a S G r tRNA7 was located at a minor region 23E on the l e f t arm of Ser chromosomG 2. The six clustered tRNA gGnes code f o r four d i f f e r e n t Ser but c l o s e l y r e l a t e d tRNA . Four of the genes specify products which Ser Ser are found in. vivo, two for tRNA^ (pDt27) and two for tRNA^ (pDtl7, pDtl6) whereas the remaining two genes (pDtl6, pDt73) would be Ser Ser transcribed to give variant products tRNA__. and tRNA..,. ° c 774 474 Table I Ser In s i t u h y b r i d i z a t i o n of plasmid DNA containing tRNA genes Plasmid No. Type of tRNA gene(s) Size of Drosophila Hind III fragment (kb) i In s i t u H ybridization s i t e pDt5 S e r 777 4.4 23E, 2L pDtl6 S e r 7 7 7 , S e r ? 7 4 6.0 12DE, X pDtl7R S e r777 3.4 12DE, X pDt27 2 S e r 4 4 4 » 4 A r § 6.4 12DE, X pDt73 S e r474 4.7 12DE, X 26 r e s p e c t i v e l y . Such variant products have not been i s o l a t e d from the Drosophila tRNA pool. These variant genes appear to be hybrids of the Ser Ser tRNA^ and tRNA^ s t r u c t u r a l genes. Cribbs adopted a t r i p l e number nomenclature system in which each number represents the Ser Ser r e l a t i o n s h i p of the variant genes to e i t h e r tRNA . or tRNA ., 4 7 genes (130). These genes contain no obvious defects which would prevent in vivo expression and are termed allogenes (Leung and Tener, personal communication). Ser Investigations of the k i n e t i c s of t r a n s c r i p t i o n of the tRNA genes and hybrid variant genes (allogenes) show that the rate of t r a n s c r i p t i o n i s a sigmoidal function of amount of DNA input. This type of dependence suggests the presence of a t r a n s c r i p t i o n i n h i b i t o r whose a c t i v i t y i s reduced by DNA input. I have shown that addition of c e r t a i n nontemplate DNAs convert the t r a n s c r i p t i o n k i n e t i c s to a hyperbolic function of DNA input which can be analyzed by Michaelis-Menten k i n e t i c s . The analysis of the reaction by these k i n e t i c s reveals that v a r i a t i o n in template a f f i n i t y f or some i n i t i a l component of the t r a n s c r i p t i o n complex and the v a r i a t i o n in the template a c t i v i t y within a preformed complex are separate from the a f f i n i t y of the o v e r a l l t r a n s c r i p t i o n complex f o r the DNA template and thus r e f l e c t independently measureable events. I have extended t h i s analysis to show that tRNA genes compete f o r formation of the t r a n s c r i p t i o n complex with c l a s s i c a l competitive i n h i b i t i o n k i n e t i c s . These k i n e t i c s allow measurement of an i n h i b i t i o n constant which relates to the a f f i n i t y of the gene for components required for t r a n s c r i p t i o n . The current state of knowledge has been summarized in a model for t r a n s c r i p t i o n of tRNA genes. 27 Materials and Methods I. General A. I s o l a t i o n of Recombinant Plasmid DNA The procedure described by Clewell and Helinski was used to i s o l a t e recombinant plasmid DNA with the following modifications (126). A single colony of bac t e r i a containing recombinant plasmid picked from a L u r i a agar plate supplemented with 50 yg/ml a m p i c i l l i n was used to innoculate 20 mis of L u r i a broth also supplemented with 50 yg/ml a m p i c i l l i n . Cultures were grown overnight at 37°C with vigorous shaking. Ten m i l l i l i t r e s of overnight culture were used to inoculate a 4 1 f l a s k containing one 1 of M9 s a l t s medium supplemented with 0.02% uri d i n e , 0.005% tryptophan, 0.005% thymidine and 0.001% thiamine. The culture was incubated at 30°C with vigorous shaking u n t i l the A... of the culture was approximately 0.6. Chloramphenicol (80 mg/ml in 95% ethanol) was added to a f i n a l concentration of 200 yg/ml and incubation of the culture continued for an a d d i t i o n a l 14 - 18 hours. The b a c t e r i a were harvested by centrifugation at 10,000xg for 15 min. at 4°C in a Sorval GS3 rotor. The b a c t e r i a l p e l l e t was resuspended in 5 mis of ice cold 25% sucrose, 10 mM T r i s - C l (pH 7.9) and 1 mM EDTA (pH 7.9) and the suspension was placed on i c e . EDTA was added to the suspension at a f i n a l concentration of 0.08 M. Afte r t h i s mixture was incubated for 10 min. on ic e , 1 ml of a f r e s h l y prepared solution of egg white lysozyme (Sigma Chemical Co.) (5 mg/ml in 10 mM T r i s - C l [pH 7.9]) was added. The suspension was c a r e f u l l y mixed by inverting the tube several times and 28 then incubated f o r an ad d i t i o n a l 20 min. on i c e . The b a c t e r i a l c e l l s were lysed by the rapid addition of 7 mis of 2% T r i t o n X-100 to the suspension. The lys a t e was centrifuged at 45,000xg for 1 hr. at 4°C in a Beckman 60 T i rotor and the supernatant f l u i d was c a r e f u l l y poured o f f . For every ml of lysate c o l l e c t e d 1.13 gms of cesium chloride were added. Afte r the s a l t completely dissolved, 0.5 mis of ethidium bromide (2 mg/ml in H 20) were added f or every 7 mis of cesium chloride s o l u t i o n . The plasmid DNA was centrifuged to equilibrium at 113,000xg for 30 hrs at 20°C in a Beckman 60 T i rotor. The band corresponding to supercoiled plasmid DNA was c a r e f u l l y removed and recentrifuged to equilibrium at 110,000 x g for 20 hrs at 20°C in a Beckman 70.1 T i rotor. The plasmid DNA was c o l l e c t e d , extracted 4 - 6 times with cesium chloride-water-saturated n-butanol, extracted once with water-saturated ether and then dialyzed overnight at 4°C against 4 l i t r e s of 10 mM T r i s - C l (pH 7.9), 1 mM EDTA (pH 7.9) and 10 mM NaCl. The plasmid DNA was analyzed by agarose gel electrophoresis to assess i t s i n t e g r i t y . B. Digestion of DNA with r e s t r i c t i o n endonucleases The reaction conditions used to cleave plasmid DNA with r e s t r i c t i o n endonucleases were as recommended by the suppliers, New England Biolabs. In general, to completely digest plasmid DNA, 1 unit of r e s t r i c t i o n enzymes per ug of plasmid DNA was added to reaction mixtures containing the appropriate buffer and incubated f o r 2-3 hours at 37°C One unit of enzyme a c t i v i t y i s defined as the amount of enzyme required to completely 29 digest 1 yg of \ DNA in 6 0 min. in a t o t a l reaction volume of 5 0 y l at 3 7°C, unless otherwise noted. C. End l a b e l l i n g of DNA DNA ( 5 - 1 0 yg) was digested with r e s t r i c t i o n endonucleases which generated fragments with 5 ' extended ends. Immediately a f t e r digestion 3 2 5 - 1 0 yCi of the appropriate [a- P]dNTP and 1 unit of Klenow fragment of E. c o l i DNA polymerase were added to the reaction mix and incubated f o r 1 0 min. at room temperature. The end-labelled DNA was loaded d i r e c t l y onto a g e l . D. Agarose gel electrophoresis Agarose gels ( 0 . 7 7 . - 2 . 0 7 , ) in TBE buffer ( 0 . 0 8 9 M Tris-Borate, 0 . 0 8 9 M Boric acid, 0 . 0 0 2 M EDTA) were used to separate, i d e n t i f y and p u r i f y DNA fragments. Bands of DNA in the gel were stained with 0 . 1 7 o ethidium bromide f o r 1 5 min. and detected by d i r e c t examination of the gel using a UV transi l l u m i n a t o r (UV Products s u p p l i e r ) . E. Polyacrylamide gel electrophoresis A stock so l u t i o n of 4 5 7 » acrylamide ( 4 3 . 5 7 o acrylamide: 1 . 5 % N,N'-methylene bisacrylamide in [w/v] H^O) was used to prepare polyacrylamide gels of the appropriate percentage. A mixture containing the required amount of 4 5 % acrylamide, TBE as buffer and 0 . 0 5 7 o ammonium persulfate (w/v) was polymerized by the addition of TEMED (N,N,N',N'-tetramethylene diamine) to a f i n a l concentration of 0 . 1 % 30 (v/v). Denaturing gels contained 7 M urea. A n a l y t i c a l and preparative gels were eit h e r 1.5 mm or 0.75 mm thick. DNA sequencing gels were 0.35 mm thick. Samples were loaded onto (non denaturing) gels using 2X TBE (0.178 M Tris-Borate, 0.178 M Boric acid, 0.004 M EDTA) 50% g l y c e r o l , 0.1% bromophenol blue and 0.1% xylene cyanol as loading buffer or onto denaturing gels using 10 M urea, 2XTBE, 0.1% bromphenol blue and 0.1% xylene cyanol as loading buffer. Radioactive samples were detected by autoradiography. Nonradioactive DNA samples were detected by fluorescence of ethidium bromide stained gels. Photographs were taken with a Polaroid MP4 camera with an orange f i l t e r . F. Autoradiography Gels containing r a d i o a c t i v e l y l a b e l l e d DNA or RNA were wrapped with p l a s t i c f i l m (Saran Wrap) then autoradiographed by overlaying a sheet of 3 o o M Hi L i t e X Ray Film i n the dark and placed at 4 C or -70 C. Exposure times varied depending on the amount of l a b e l incorporated. The X ray f i l m was developed according to the manufacturer's i n s t r u c t i o n s . For extra s e n s i t i v i t y an i n t e n s i f y i n g screen (Dupont Cromex Lightning - plus; 126) was placed on top of the X ray f i l m and the autoradiograph was o exposed at -70 C. DNA sequencing gels were drie d onto Whatman 3 MM f i l t e r paper using a Bio-Rad Slab Gel Drier Model 1125B p r i o r to autoradiography. 31 G. I s o l a t i o n of nucleic acid from polyacrylamide s e l s : E l e c t r o e l u t i o n Desired DNA fragments were i d e n t i f i e d on preparative polyacrylamide gels by ethidium bromide staining or autoradiography. The gel s l i c e containing the DNA fragment was excised from the g e l . The gel s l i c e was chopped into f i n e pieces and placed into an e l e c t r o e l u t i o n tube containing 80 mM Tris-Acetate (pH 7.0). E l e c t r o e l u t i o n was c a r r i e d out for 6 hrs at 3 mA/tube. The eluate containing the desired DNA fragment was removed from the d i a l y s i s bag and extracted once with water saturated phenol. The r e s i d u a l phenol remaining in the aqueous solution was removed by 3 extractions of water saturated ether. The DNA was p r e c i p i t a t e d by the addition of 1/10 volume of 3 M sodium acetate (pH 8.5) and 3-4 volumes of 95% ethanol. The DNA was dissolved in 200 y l of TE (10 mM T r i s - C l pH 7.9 and 1 mM EDTA) and r e p r e c i p i t a t e d as above. The p e l l e t containing the DNA fragment was dried b r i e f l y under vacuum then resuspended in a small volume of TE (pH 7.9). H. I s o l a t i o n of nucleic acid from agarose gels: Trough e l u t i o n R e s t r i c t i o n fragments were recovered from ethidium bromide stained preparative agarose gels by electrophoresis of the DNA into a trough f i l l e d with TBE buffer which had been cut out of the gel d i r e c t l y i n front of the leading edge of the band, for approximately 20 min. The DNA recovered was extracted 3 times with n-butanol to remove the ethidium bromide followed by one extraction with ether. The DNA was p r e c i p i t a t e d 32 twice with ethanol as described in section G. The r e s u l t i n g p e l l e t was dissolved in an appropriate volume of TE buffer (pH 7.9). I. Synthesis of [5'- 3 2P]pCp 32 [5'- P]pCp was prepared by incubating 1.22 mM Cp (2' and 3*), 10 mM T r i s - C l (pH 9), 10 mM MgCl 2 > 10 mM DTT, 5 uM ATP, 30 uCi 32 ["Y- P]ATP (S.A.> 5000 Ci/nmole, Amersham) and 4 units of T4 polynucleotide kinase (P.L. Biochemicals) in a 10 y l volume f o r 90 min. at 37°C. The mixture was heated for 1 min. in a b o i l i n g water bath to 32 terminate the reaction. The [5'- P]pCp was used as substrate to end Ser l a b e l tRNA^ without further p u r i f i c a t i o n . J. 3' End l a b e l l i n g of tRNA 32 Newly synthesized [5'- P]pCp was incorporated into the 3' end of Ser the tRNA7 by using RNA l i g a s e as described by England and Uhlenback (127). The amount of incorporated nucleotide was determined by p r e c i p i t a t i n g 1 y l and 3 y l aliquots of the reaction. The aliquots were d i l u t e d in 300 y l of 10 mM T r i s - C l (pH 7.9), 1 mM EDTA (pH 7.9), 10 mM NaCl and 0.5 mg/ml yeast RNA. The RNA was p r e c i p i t a t e d by adding 1 ml of 10% T r i c h l o r o a c e t i c Acid. The radioactive tRNA was allowed to p r e c i p i t a t e f or 5 min. at room temperature p r i o r to f i l t r a t i o n through a glass f i b r e f i l t e r . The f i l t e r s were drie d and then the r a d i a t i o n was detected counted using s c i n t i l l a t i o n f l u i d containing 1% diphenyloxazole in toluene in a Beckman LS 7000 S c i n t i l l a t i o n Counter. The r a d i o a c t i v e l y 33 l a b e l l e d tRNA was used as a hy b r i d i z a t i o n probe and a molecular weight marker on polyacrylamide gels containing in v i t r o t r a n s c r i p t i o n products. K. Southern trans f e r of DNA Afte r electrophoresis of r e s t r i c t i o n fragments on agarose gels, the gel was stained with ethidium bromide and photographed using a Polaroid MP 4 camera. The DNA was denatured and transferred onto n i t r o c e l l u l o s e as described by Southern (128). L. Hybridization of [5'- 3 2P]pCp l a b e l l e d tRNA Hybridization of l a b e l l e d tRNA to DNA immobilized onto n i t r o c e l l u l o s e was as performed by Southern except that the prehybridization and hybr i d i z a t i o n buffer contained 0.6 M NaCl, 0.06 M sodium c i t r a t e , 20% formamide (v/v), 0.5% sodium dodecyl su l f a t e and 0.02% bovine serum albumin. M. Plasmid DNA Ser Recombinant plasmids shown to contain tRNA genes were i s o l a t e d as f i r s t described by Dunn et a l . (129). Further c h a r a c t e r i z a t i o n and See sequence determination of the tRNA genes were reported by Cribbs (130) and Newton (139) . N. Treatment of 3' extended ends with T4. DNA polymerase The 3' extended ends r e s u l t i n g from cleavage of pDt 16 DNA with Hha I r e s t r i c t i o n endonuclease were removed by t r e a t i n g the DNA fragments with 4 34 units of T DNA polymerase (Boehringer Mannheim) in the presence of 0.5 mM dCTP and 0.5 mM dGTP using 6 mM T r i s - C l (pH 7.4), 50 mM NaCl, 6 mM MgCl^ and 6 mM B-mercaptoethanol as buffer at 30°C for 15 min. The T^ DNA polymerase was inactivated by heating to 65°C for 15 min. 0. Preparation of l i n e a r blunt ended pUC8 The plasmid pUC8 (20 yg) was l i n e a r i z e d with 10 units of Smal in a 20 y l reaction mixture. A f t e r 4 hrs of incubation at 37°C in the appropriate buffer, the r e s t r i c t i o n enzyme was inactivated by heating the reaction vessel at 65°C for 15 min. A 1 yg aliquot of l i n e a r i z e d plasmid DNA was electrophoresed on a 0.7% agarose gel to assess the extent of digestion. The l i n e a r i z e d pUC8 was stored in TE buffer at -20°C u n t i l required. P. L i g a t i o n of DNA r e s t r i c t i o n fragments to l i n e a r i z e d pUC8 Ligation reactions (10 y l ) containing 0.3 yg of l i n e a r i z e d pUC8, 50 mM T r i s - C l (pH 7.8), 10 mM MgCl 2 > 20 mM d i t h i o t h r e i t o l , 1 mM ATP, 50 yg/ml bovine serum albumin and 8 units of T DNA l i g a s e (New England Biolabs) were c a r r i e d out with 3 d i f f e r e n t concentrations of i n s e r t DNA. For blunt end l i g a t i o n s a 1:1, 5:1, and a 10:1 molar r a t i o of i n s e r t to vector DNA was used. When l i g a t i n g DNA fragments containing extended ends a 1:1, 2:1, and a 3:1 molar r a t i o was used. The mixtures were incubated at 12-16°C overnight. Modifications of t h i s procedure w i l l be noted in the r e s u l t s . 35 Q. Transformation of E. c o l i (JM 83) E. c o l i (JM 83) was transformed with l i g a t e d DNA as described by Messing et a l . (131) with some modifications. F i f t y m i l l i l i t r e s of 2xYT media (16 gms tryptone, 10 gms yeast extract, 10 gms NaCl) was inoculated with 0.5 mis of an overnight b a c t e r i a l culture of E. c o l i (JM 83) grown on the same media. The c e l l s were grown with vigorous shaking at 37°C f or 3 hrs. The ba c t e r i a culture was cooled on ice for 5 min. p r i o r to harvesting. A f t e r discarding the supernatant, the c e l l s were resuspended in 25 mis of a ice cold s t e r i l e solution of 50 mM C a C l 2 and 10 mM T r i s - C l (pH 8.0). The c e l l suspension was placed i n an ice bath f o r 20 min. and then centrifuged. The c e l l p e l l e t was resuspended in 3.5 mis of ice cold 50 mM C a C l 2 , 10 mM T r i s - C l (pH 8.0) solution and placed on ice for 1 hr. Aliquots (0.1 ml) of competent c e l l s were slowly pipetted into the p r e - c h i l l e d 1.9 ml Eppendorf tubes containing 10 y l of l i g a t e d DNA (Section P). The DNA-cell suspension was incubated on ice for an hour followed by a 3 min. heat shock treatment at A2°C. To each tube 1.0 ml of 2xYT media was added and the mixture was incubated at 30°C f or 1 hr in a shaking water bath. R. Selection of cloned fragments Af t e r incubation for 1 hr at 30°C, 100 y l of the DNA-cell suspension was f i r s t mixed with 50 y l of X-gal [2% (w/v) 5-bromo-4-chloro-3-indolyl-B-D-galactoside (Sigma Chemical Co.) in dimethyl formamide] then plated f o r i n d i v i d u a l colonies on 2xYT agar plates containing 50 yg/ml a m p i c i l l i n . Bacteria containing recombinant 36 plasmids were selected f o r by t h e i r resistance to a m p i c i l l i n and t h e i r i n a b i l i t y to metabolize X-gal. White colonies were picked i n a f i e l d of blue colonies. The e f f i c i e n c y of transformation was tested each time by using a) supercoiled pUC8 DNA, to assess transformation frequency, b) l i g a t e d Smal cut pUC8 DNA, to monitor the r e l i g a t i o n reaction and c) unligated Smal cut pUC8 DNA, to determine the frequency of transformation of l i n e a r i z e d DNA. The frequency of transformation with l i g a t e d DNA varied over the 3 range of 2 - 10 x 10 white, a m p i c i l l i n r e s i s t a n t colonies per ug of l i g a t e d DNA. White, a m p i c i l l i n r e s i s t a n t colonies were picked and spotted onto 2xYT agar plates containing 50 ug/ml a m p i c i l l i n and onto n i t r o c e l l u l o s e f i l t e r s placed on 2xYT agar plates also containing 50 yg/ml a m p i c i l l i n . The c e l l s were grown overnight at 37°C and the f i l t e r s were treated according to the method of Grunstein and Hogness (132) to f i x the plasmid Ser DNA. The f i l t e r s were hybridized with 3' end l a b e l l e d tRNA^ as described i n Section K. S. Rapid small scale i s o l a t i o n of plasmid DNA This procedure was performed as described by Holmes and Quigley (133). Tubes containing 3-5 mis of L u r i a Broth supplemented with 50 yg/ml a m p i c i l l i n were inoculated with b a c t e r i a containing recombinant plasmids and grown overnight at 30°C with vigorous aeration. An aliquot (1.9 ml) of the culture was subjected to 30 seconds of ce n t r i f u g a t i o n i n an Eppendorf centrifuge at maximum speed. The culture media was discarded 37 leaving the b a c t e r i a l p e l l e t as dry as possible. The p e l l e t was resuspended in 350 y l of solution containing 8.0% sucrose, 0.5% T r i t o n X-100, 50 mM EDTA (pH 8.0) and 10 mM T r i s - C l (pH 8.0) by vortexing. The suspension was then treated with 25 y l of egg white lysozyme [10 mg/ml in 10 mM T r i s - C l (pH 8.0)], mixed, then placed in a b o i l i n g water bath f o r exactly 40 seconds. The lysate was removed from the water bath and immediately centrifuged for 15 min. at maximum speed i n an Eppindorf centrifuge. The p e l l e t s were removed using s t e r i l e toothpicks. The nucleic acid i n the lysate was p r e c i p i t a t e d by the addition of 1/10 volume of sodium acetate (pH 8.5) and 1 volume of isopropyl alcohol. The mixture was stored i n a dry ice/ethanol bath f o r 30 min. p r i o r to ce n t r i f u g a t i o n for 8 min. i n an Eppendorf centrifuge. The supernatant s o l u t i o n was discarded. The p e l l e t was dried i n a dessicator then dissolved in 50 y l of TE buffer (pH 7.9). Usually 10 - 20 y l of t h i s s o l u t i o n was treated with a desired r e s t r i c t i o n enzyme for 2 - 3 hours under the appropriate reaction conditions. DNase free RNase (Sigma) was added to the r e s t r i c t i o n enzyme digest at a f i n a l concentration of 30 yg/ml and o incubated for an ad d i t i o n a l 20 min at 37 C. The DNA fragments were analyzed by gel electrophoresis using r e s t r i c t e d vector DNA as a size standards. Small plasmid preparations (250 ml) were made from a p o s i t i v e colonies according to the method described e a r l i e r (Section A) except that a l l volumes were reduced by one-fourth. 38 T. P u r i f i c a t i o n of plasmid DNA for DNA sequence analysis The nucleotide sequence of DNA cloned in to pUC8 plasmids was determined by using the Messing procedure (134) as modified by Dr. Swee Han Goh and Gary O ' N e i l l . To determine the nucleotide sequence of plasmid DNA by using the dideoxy chain terminator sequencing method i n i t i a l l y described by Sanger et a l . , (135,136), i t i s e s s e n t i a l that the DNA be free from contaminating RNA. Plasmid DNA p u r i f i e d by two cesium chloride density gradients could not be subjected to sequencing as such and had to be further p u r i f i e d . Plasmid DNA was prepared for sequencing by incubating 20 yg of DNA in 100 y l of TEN buffer [10 mM T r i s - C l (pH 7.9), 1 mM EDTA (pH 7.9) and 10 mM NaCl] containing 20 yg/ml DNase free RNase for 1 hr at 37°C. The plasmid DNA solution was d i l u t e d to 0.3 mis with TEN buffer, layered on top of a centrifuge tube containing 1 M NaCl plus TE buffer and centrifuged for 6 hrs in a Beckman SW 50.1 rotor at 150,000xg at 20°C. The supernatant was discarded and the p e l l e t containing plasmid was redissolved in 300 y l of TE buffer. The p u r i f i e d plasmid DNA was p r e c i p i t a t e d with ethanol twice, resuspended with 20 y l of TE buffer then digested with either EcoRI or H i n d l l l r e s t r i c t i o n endonucleases in the appropriate buffers depending on whether the forward or the reverse sequencing reactions were performed. U. DNA sequence analysis Reaction mixtures of 12.5 y l volumes containing 7 mM T r i s - C l (pH 7.9), 50 mM NaCl and 7 mM MgCl 2 as buffer, 1 yg of plasmid DNA which has been completely digested with EcoRI on H i n d l l l r e s t r i c t i o n 39 endonucleases and 40 ng of either forward primer (used with EcoRI digested template) or reverse primer (used with H i n d l l l digested template) were heated to 90°C for 5 min. and r a p i d l y cooled for 5 min. in an ice-water bath to anneal primer DNA to the template DNA. Af t e r completion of the 32 annealing reaction, 10 yCi of [a- P]dATP (> 3000 Ci/mmole, New England Nuclear), 1 y l of 15 yM dATP, 1 y l of 0.1 M d i t h i o t h r e i t o l and 4 units of Klenow DNA polymerase were added d i r e c t l y to the mixture. Aliquots of 3 y l each were removed from t h i s mixture, added to 4 reaction tubes containing 2 y l of the appropriate dideoxy nucleoside triphosphate/deoxynucleoside triphosphate mix and incubated at 24°C for 15 min. The concentrations of the dideoxy nucleoside mix was as described (134). One y l of a cold chase solution containing 0.3 mM dATP, dTTP, dCTP and dGTP was added to each of the four reaction tubes and allowed to incubate at 24°C f o r an a d d i t i o n a l 15 min. The reactions were terminated by the addition 10 y l of 90% formamide containing 0.1% xylene cyanol and 0.1% bromophenol blue. The samples were denatured in a b o i l i n g water bath for 5 min. p r i o r to loading onto an 8%-7 M urea DNA sequencing gel. A f t e r 2 hours of electrophoresis at 1200 V (30 V/cm) the same sequencing reactions were applied to adjacent s l o t s in the polyacrylamide gel and electrophoresis continued for an a d d i t i o n a l 2 hours. The gel was drie d onto a Whatman 3 MM f i l t e r paper and autoradiographed overnight (Section F ) . 40 V. In v i t r o t r a n s c r i p t i o n and analysis of RNA products The S100 extracts used f o r t r a n s c r i p t i o n were prepared from Drosophila Schneider II c e l l s as described by Rajput et a l . , (137). A l l in v i t r o t r a n s c r i p t i o n reactions had a f i n a l volume of 50 y l and contained 19 mM Tris-HCl (pH 7.9); 110 mM KCI, 7 mM MgCl 2, 3 mM d i t h i o t h r e i t o l , 2.5 y/ml a-amanitin (Boerhinger Mannheim), 6.5 u/ml creatine phosphokinase, 5 mM creatine phosphate, 600 uM each of the 32 unlabelled ribonucleoside triphosphates, 25 yM [a- P]UTP (3-5 Ci/mmole), 1 ng to 1 yg of template DNA, 0 to 1 yg pBR322 DNA and 25 y l of Drosophila S100 extract. Transcription reactions were i n i t i a t e d by addition of the S100 extract to a l l other components and were c a r r i e d o out for 90 min at 23 C. The reactions were terminated by the addition of 50 y l of 1 M sodium acetate (pH 4.6). The mixture was extracted with 75 y l of phenol e q u i l i b r a t e d with 0.05 M sodium acetate (pH 4.6) and the aqueous phase was p r e c i p i t a t e d with ethanol. The t r a n s c r i p t i o n products were separated by electrophoresis through 10% polyacrylamide gels containing 7 M urea and TBE as a buffer. The gel was autoradiographed to l o c a l i z e the t r a n s c r i p t i o n products and the radioactive bands were excised from the gel for quantitation of the amount of product by Cerenkov counting. 41 Results I. Construction of recombinant plasmids A. pUC8 as a cloning vector The plasmid pUC8 was constructed as a cloning vector by Viera and Messing (131). The construction consists of the o r i g i n of r e p l i c a t i o n and 6 lactamase gene from the plasmid pBR322 fused to the a fragment of the 6 galactosidase gene which contains 9 unique r e s t r i c t i o n enzyme s i t e s as a pol y l i n k e r i n the amino portion of B galactosidase. There are two advantages to using pUC8 as a cloning v e h i c l e ; 1) i n s e r t i o n of DNA fragments into any one of the unique r e s t r i c t i o n enzyme s i t e s in the pol y l i n k e r can be p o s i t i v e l y selected on the basis of a m p i c i l l i n resistance and lack of color production on an indicato r plate and 2) synthetic universal DNA primers complementary to eit h e r strand of the po l y l i n k e r can be used to perform rapid "dideoxy" chain terminator sequencing of the border sequences of cloned fragments inserted into the pol y l i n k e r (135) . E. c o l i JM83 and the plasmid pUC8 were a generous g i f t from Dr. Steve Wood. Large scale preparations of pUC8 were performed as in the Materials and Methods. pUC8 was prepared f o r blunt end digestion reactions by tr e a t i n g 20 ug of the plasmid DNA with 20 units of Smal in the appropriate buffer f o r 5 hours at 37°C, i n a t o t a l volume of 50 y l . The Smal l i n e a r i z e d DNA was heated to 65°C f o r 20 min. to inacti v a t e the r e s t r i c t i o n endonuclease, d i l u t e d to 0.3 mg/ml by adding TE buffer, then stored at -20°C u n t i l required. 42 Figure 1. P a r t i a l r e s t r i c t i o n endonuclease map of the sequenced portion Ser of pDtl6 containing two tRNA genes. The A l u l and Hhal r e s t r i c t i o n endonuclease map was constructed from the DNA sequence of pDtl6. Hybridization analysis suggested two 510 bp A l u l DNA fragments (B) and two 980 bp Hhal DNA fragments (C) would contain Ser the i s o l a t e d tRNA genes (see Figure 2). The transfer RNA genes are Ser shown by an open box while the numbers below denote the type of tRNA gene. Flanking DNA sequences are shown by the s o l i d l i n e s . o. n U o 00 0> a . JO o oo 44 B. Subcloning of two tRNA genes contained in pDt!6 Ser Plasmid, pDtl6 was shown to contain two tRNA genes, a Ser Ser tRNAj 7 7 gene and a tRNA^^ gene separated by 375 bp (130). Examination of the DNA sequence of pDtl6 revealed two unique r e s t r i c t i o n enzyme s i t e s which could be used to subclone the two genes, an A l u l s i t e , Ser S e r 339 bp 3' to the tRNA,,, gene and 36 bp 5' to the tRNA,,. 777 774 Ser gene and a Hhal s i t e , 70 bp 3' to the tRNA^^ gene and 305 bp 5* to Ser the tRNA^^ (Figure 1). The l o c a t i o n of a second A l u l s i t e 92 bp Ser 5' to the tRNA^^^ gene indicates that digestion of pDtl6 with A l u l Ser would r e s u l t in a 510 bp DNA fragment containing the tRNA^^^ gene. The siz e of other DNA fragments expected to contain e i t h e r tRNA gene could not be determined since information about the l o c a t i o n of other A l u l and Hhal s i t e s was not a v a i l a b l e . Ser DNA fragments which contain i s o l a t e d tRNA genes were i d e n t i f i e d as follows. DNA fragments generated by the digestion of 5 ug of pDtl6 with e i t h e r 7 units of A l u l or 7 units of Hhal in r e s t r i c t i o n enzyme buffer at 37°C for 4 hours, were separated by electrophoresis on a 1.8% agarose gel containing 0.1% ethidium bromide. Af t e r completion of electrophoresis, the gel was photographed (Figure 2). DNA in the gel was denatured, transferred onto n i t r o c e l l u l o s e f i l t e r s and then hybridized Ser with 3' end l a b e l l e d tRNA ^ as described in the Materials and Methods. A f t e r h y b r i d i z a t i o n , the f i l t e r was washed and autoradiographed o overnight at 4 C. The r e s u l t i n g autoradiography (Figure 2) showed that a single 510 bp DNA fragment generated by A l u l and a single 980 bp DNA fragment generated 45 Figure 2. Determination of the A l u l and Hhal r e s t r i c t i o n endonuclease Ssr fragments which contain a tRNA gene. The plasmid, pDtl6 (5 yg) was digested with the r e s t r i c t i o n endonucleases A l u l (lane 2) and Hhal (lane 4), the r e s u l t i n g fragments were electrophoresed on a 1.8% agarose gel containing ethidium bromide and the gel was photographed. DNA in the gel was denatured, transferred to Ser n i t r o c e l l u l o s e , hybridized to 3' end l a b e l l e d tRNA^ and analyzed as described in the Materials and Methods. A 510 bp A l u l fragment (lane 5) Ser and a 980 bp Hhal fragment (lane 6) hybridized the tRNA^ probe. Lane 1 shows pBR322 DNA digested with A l u l while lane 4 shows the same DNA digested with Hhal. These DNAs were used as molecular weight markers (143). The arrows point to the 510 bp A l u l fragment in lane 2 and the 980 bp Hhal fragment in lane 3. The f a i n t band in lane 5 i s due to the probe hy b r i d i z i n g to a p a r t i a l l y digested DNA fragment. 46 1 2 3 4 5 6 Se r by Hhal harbored a tRNA gene. From the sequence data av a i l a b l e f o r pDtl6, I expected to see two A l u l DNA fragments hybr i d i z i n g the tRNA probe: one of 515 bp in length and another of a minimum length of 260 bp, and two Hhal DNA fragments; one greater than 550 bp and the other greater Ser than 310 bp, which would hybridize the tRNA 7 probe. The Ser hy b r i d i z a t i o n of the l a b e l l e d tRNA 7 to a single A l u l DNA fragment and a single Hhal fragment indicated that e i t h e r the procedure outlined was not s e n s i t i v e enough to detect the smaller DNA fragments which Ser possibly obtained a tRNA gene but were not bound to the n i t r o c e l l u l o s e f i l t e r s or that there were two 510 bp A l u l fragments and Ser two 980 bp Hhal DNA fragments which contained the i s o l a t e d tRNA genes. Assuming the l a t t e r to be true, I proceeded to subclone the 510 bp A l u l and 980 bp Hhal DNA fragments. DNA was prepared for subcloning by digesting 40 ug of pDtl6 with eit h e r 40 units of Hhal or 40 units of A l u l i n the appropriate buffer to 6 hours at 37°C. P r i o r to electrophoresis through a 1.8% agarose g e l , the 3' extended ends on the Hhal DNA fragments were made into blunt ends by using the 3* exonuclease a c t i v i t y of T^ DNA polymerase. The pDtl6 DNA cleaved with Hhal was treated with 2 units of T^ DNA polymerase f o r 15' at 37°C in Hhal r e s t r i c t i o n enzyme buffer containing 2 mM of a l l 4 dNTPs. The 3'-* 5* exonuclease a c t i v i t y of T^ , DNA polymerase which i s considerably more active on single stranded DNA than on double stranded DNA removes the protruding ends (138). The exonuclease a c t i v i t y i s blocked on double stranded DNA by the 5' -» 3* polymerase a c t i v i t y . No 48 processing of the A l u l DNA fragments, which already contained blunt ends, was required. The 510 bp A l u l and the 980 bp Hhal DNA fragments were eluted from the agarose gel as described i n the Materials and Methods. The eluate containing DNA was extracted 3 times with water saturated n-butanol to remove the ethidium bromide. Residual butanol in the aqueous phase was removed by one extraction with water saturated ether. The DNA was pr e c i p i t a t e d twice with ethanol, dr i e d in vacuo, and then resuspended i n 20 y l of TE buffer. A 3 y l aliquot of the eluted 510 bp A l u l DNA fragment and the 980 bp Hhal DNA fragment along with 1 yg of A l u l digested pDtl6 was subjected to agarose gel electrophoresis. The amount of DNA recovered by e l e c t r o e l u t i o n was approximated by comparing the UV fluorescence of each DNA band. To subclone the i s o l a t e d blunt ended 510 bp A l u l and the 980 bp Hhal DNA fragments, 0.3 yg of Smal l i n e a r i z e d pUC8 DNA was mixed with 3, 5 or 10 molar excess of either DNA fragment i n 10 y l reaction volumes containing 50 mM T r i s - C l (pH 7.9), 10 mM MgCl 2 > 20 mM d i t h i o t h r e i t o l , 1 mM ATP and 50 yg/ml bovine serum albumin as l i g a s e buffer and 10 units of T^ DNA l i g a s e (New England Biolabs). The l i g a t i o n reactions were incubated overnight at 16°C. As a con t r o l , the l i g a t i o n of 0.3 yg of Smal l i n e a r i z e d pUC8 DNA in the absence of DNA fragments was performed. The l i g a t e d DNA was transformed into E. c o l i JM83 as described in the Materials and Methods. Bacteria harboring recombinant plasmids which were selected by t h e i r resistance to 50 yg/ml a m p i c i l l i n and t h e i r i n a b i l i t y to metabolize X-gal, were picked and transferred to a master plate 49 containing 2xYT and 50 yg/ml a m p i c i l l i n . Bacteria from the master plate were transferred to a n i t r o c e l l u l o s e f i l t e r which was placed on top of a 2xYT plate containing 50 yg.ml a m p i c i l l i n and were grown at 37°C overnight. The colonies on the n i t r o c e l l u l a s e f i l t e r s were lysed and the l i b e r a t e d DNA was f i x e d to the f i l t e r by the method of Grunstein and Hogness (132). The DNA on the f i l t e r s was hybridized with 3* end l a b e l l e d Ser tRNA 7 as described in the Materials and Methods. A f t e r h y b r i d i z a t i o n , the f i l t e r was monitored by autoradiography. Approximately 60% of the b a c t e r i a selected as containing recombinant plasmids with A l u l Ser DNA inserts were found to contain a tRNA gene. However, only 35% of the b a c t e r i a picked as containing recombinant plasmids constructed with See Hhal DNA fragments were found to contain a tRNA gene. To analyze the structure of the recombinant plasmids which were found Ser to contain a tRNA gene, a rapid, small scale i s o l a t i o n of plasmid DNA was prepared from 6 b a c t e r i a l colonies containing plasmids with A l u l DNA Ser inserts complimentary to the tRNA probe and from 6 colonies Ser containing plasmids with Hhal DNA inserts complimentary to the tRNA probe as described in the Materials and Methods. A 10 y l aliquot of i s o l a t e d plasmid DNA was digested with 8 units of EcoRI and 8 units of H i n d l l l in H i n d l l l r e s t r i c t i o n enzyme buffer f or 3 hours at 37°C. The digested DNA was treated with 20 yg/ml of DNase free RNase for 10 min. o at 37 C before electrophoresis on a 1.4% agarose g e l . The size of the cloned DNA fragments were determined. Five out of the six recombinant plasmids i s o l a t e d from both the A l u l subclones and the Hhal subclones were found to contain the 510 bp and the 980 bp inserts r e s p e c t i v e l y . Large 50 Figure 3. Analysis of the pDtl6 subclones by r e s t r i c t i o n endonuclease cleavage. The f i v e plasmids containing the 980 bp Hhal fragment were digested with A l u l ; pDtH16#l (lane 5), pDtH16#2 (lane 4), pDtH16#3 (lane 3), pDtH16#4 (lane 2) and pDtH16#5 (lane 1). The f i v e plasmids containing the 510 bp A l u l fragment were digested with Hhal; pDtA16-2#l (lane 11), pDtA16-2#2 (lane 10), pDtA16-2#3 (lane 9), pDtA16-2#4 (lane 8) and pDtA16-2#5 (lane 7). The r e s u l t i n g DNA fragments were electrophoresed on a 5% polyacrylamide g e l , stained with 0.1% ethidium bromide and the gel was photographed. Lane 6 shows pUC8 DNA cleaved with A l u l as molecular weight marker. The size (bp) of ins e r t DNA fragments i s shown on the side of the g e l . 51 bp 1 2 3 4 5 6 7 8 9 1 0 1 1 bp 530-380-290--.840 -.460 -.380 55MMMH* 52 scale plasmid preparations from one 1 cultures were performed as described in the Materials and Methods on the 5 A l u l subclones (pDtA16-2#l-5) and the 5 Hhal subclones (pDtH16#l-5) which were found to contain the appropriate si z e i n s e r t . To determine whether a l l subclones derived from the A l u l or the Hhal DNA fragments were i d e n t i c a l approximately 2 yg of the plasmids pDtA16-2#l-5 were treated with 4 units of Hhal and 2 yg of the plasmids pDtH16#l-5 were treated with 4 units of A l u l f o r 3 hours at 37°C in the appropriate buffer. The r e s u l t i n g fragments were electrophoresed on a 5% polyacrylamide g e l , stained with 0.1% ethidium bromide an then analyzed by Sex* fluorescence. I f the two tRNA genes were separated onto two 510 bp A l u l DNA fragments and two 980 bp Hhal DNA fragments then I would expect Ser the 510 bp A l u l DNA fragments which contained the tRNA ? 7 7 gene to be cleaved with Hhal and the 510 bp A l u l DNA fragment which contained the Ser tRNA 7 7 7 gene to be r e s i s t a n t to Hhal digestion (see Figure 1 for d e t a i l s ) . As shown in Figure 3 the digestion of the plasmids pDtA16-2#l-5 with Hhal showed two d i s t i n c t r e s t r i c t i o n endonuclease fragment patterns. The Hhal DNA fragments generated from pDtA16-2#l (lane 11) were c l e a r l y d i f f e r e n t from the other 4 plasmids pDtA16-2#2-5 (lanes 7-10). These plasmids a l l yi e l d e d i d e n t i c a l r e s t r i c t i o n endonuclease fragment patterns on the g e l . The 840 bp Hhal DNA fragment r e s u l t i n g from the digestion of pDtA16-2#2-5 were consistent with the subcloning of the 510 bp A l u l Ser fragment containing the tRNA 7 7^ gene regardless of o r i e n t a t i o n (Figure 4c and d). Ser Figure 4. Possible orientations of the tRNA genes on the 510 bp A l u l DNA fragments. a. and b. show the two possible orientations of the subcloned Ser tRNhjjj gene in pUC8 while c. and d. show the two possible Ser orientations of the subcloned tRNA^^^ gene. The A l u l DNA fragment i s shown by an open box. The pUC8 DNA sequences surrounding the in s e r t are shown by s o l i d l i n e s . The closed Ser boxes depict the tRNA genes while the small arrows below these boxes show the d i r e c t i o n of t r a n s c r i p t i o n . The r e s t r i c t i o n endonucleases EcoRI and H i n d l l l cleave at unique s i t e s in pUC8 and are used to prepare the template DNA for dideoxy DNA sequencing. The r e s t r i c t i o n endonuclease Hhal cleaves pUC8 at many s i t e s and once in the A l u l fragment containing Ser the tRNA 7 7 7 gene. The thick arrows show the d i r e c t i o n of DNA sequencing. HhaI EcoR I HhaI Hha I Reverse primer —+-100 bp Hind III Hha I tRNA g e n e - *— Forward primer l i t R N A f ® ' gene 55 Cleavage of pDtA16-2#l with Hhal yielded two DNA fragments derived from the cloned i n s e r t , a 460 bp and a 380 bp DNA fragment (approximate s i z e ) . The 460 bp and 360 bp Hhal DNA fragments were the size of DNA fragments which would be expected from the cleavage of the 510 bp A l u l Ser containing the tRNA^^ gene cloned into the Smal s i t e of pUC8 in the o r i e n t a t i o n described by Figure 4a. Analysis of A l u l digested pDtH16#l-5 subclones was not as cl e a r because of the many A l u l cleavage s i t e s in the 980 bp Hhal DNA i n s e r t s . However, A l u l digestion of the pDtH26 subclones (Figure 3) d i d reveal two d i f f e r e n t r e s t r i c t i o n endonuclease fragment patterns consistent with the subcloning of two Hhal DNA fragments of i d e n t i c a l s i z e . The cloned inserts in the 4 plasmids, pDtH16#2-5 (lanes 1-4) were cleaved with A l u l at 4 locations y i e l d i n g , 380 bp, 265 bp, 165 bp and 155 bp DNA fragments. Digestion of pDtH16#l (lane 5) with A l u l r e s u l t e d i n 3 DNA fragments derived from the cloned i n s e r t , a 530 bp, a 290 bp, and a 170 bp fragment. The production of a 265 bp A l u l DNA fragment from the 4 plasmids, pDtH16#2-#5 would be expected i f the 980 Ser bp Hhal i n s e r t contained the tRNA^^ gene. The 290 bp and 530 bp A l u l DNA fragments generated from pDtH16#l treated with A l u l i s consistent with the expected DNA fragments i f the 980 bp Hhal i n s e r t contained the Ser tRNA^^^ gene (See Figure 1). To confirm the i d e n t i t y of the subcloned DNAs, the plasmids pDtA16-2#l and #3 and the plasmids pDtH16#l and #5 were subjected to dideoxy chain termination DNA sequencing (135). Because the dideoxy chain terminator DNA sequencing method depends on a DNA primer to i n i t i a t e DNA synthesis then i t i s absolutely e s s e n t i a l that plasmid DNA preparations be 56 free from contaminating RNAs which could p o t e n t i a l l y cause f a l s e priming. Although plasmid DNA was p u r i f i e d by two CsCl density gradients, i t was s t i l l necessary to p u r i f y i t further. Residual RNA was removed from DNA preparations by treatment with DNase free RNase A p r i o r to c e n t r i f u g a t i o n of the DNA on a 1 M NaCl gradient (see Materials and Methods). Af t e r completion of DNA p u r i f i c a t i o n , the plasmids were resuspended in 40 u l TEN buffer and s p l i t into two, 20 y l ali q u o t s . Plasmid DNA in one 20 y l aliquot was prepared for DNA sequencing in the forward d i r e c t i o n by treatment with 8 units of EcoRI at 37°C for 3 hours. To sequence the DNA in the reverse d i r e c t i o n plasmid DNA was l i n e a r i z e d with 8 units of H i n d l l l at 37°C for 3 hours. A 2 y l aliquot of each preparation of l i n e a r i z e d DNA was electrophoresed on a 0.7% agarose gel to assess the l e v e l of DNA digestion and to approximate the concentration of l i n e a r i z e d DNA. I have found that complete digestion of plasmid DNA was necessary for e f f i c i e n t priming of the DNA sequence reaction. DNA sequence reactions containing the appropriate primer annealed to l i n e a r i z e d plasmid template were c a r r i e d out as described in the Materials and Methods. The DNA sequence analysis of the 4 plasmids, pDtA16-2#l (Figure 5, 6), pDtA16-2#3 (Figure 7, 8), pDtH16#l (Figure 9, 10) and pDtH16#5 (Figure Ser Ser 11, 12) confirmed that the tRNA^^ gene and the tRNA^^ gene were i s o l a t e d on two 510 bp A l u l DNA fragments and two 980 bp Hhal DNA fragments. The plasmids pDtA16-2#l and pDtH16#5 contain the Seir tRNA 7 7^ gene while the plasmids pDtA16-2#3 and pDtH16#l contain the tRNA 7 7 7 gene. The DNA sequence of the cloned inserts in pDtA16-2#l in both the forward (Figure 5) and reverse d i r e c t i o n (Figure 6) and the 57 Figure 5. DNA sequence analysis of pDtA16-2#l: A Forward d i r e c t i o n The DNA sequence of the subcloned A l u l DNA fragment was determined by the method of Viera and Messing (131) as modified in the Materials and Methods. Lanes 1-4 and lanes 5-8 represent forward sequencing reactions s p e c i f i c f o r A, C, G and T respectively. The samples in lanes 5-8 were electrophoresed 2 hours longer than the samples in lanes 1-4. The DNA sequence i s shown on the side of the autoradiograph. The sequence i s i d e n t i c a l to the coding strand of the 510 bp A l u l DNA fragment containing Sexr the tRNA 7 7 gene as determined by Cribbs (130). 58 59 Figure 6. DNA sequence analysis of pDtA16-2#l: B. Reverse d i r e c t i o n . The DNA sequence of the subcloned A l u l DNA fragment was determined by the method of Viera and Messing (131) as modified in the Materials and Methods. Lanes 1-4 represent reverse sequencing reactions s p e c i f i c f o r A, C, 6 and T re s p e c t i v e l y . The DNA sequence i s shown on the side of the autoradiograph. The sequence i s i d e n t i c a l to the noncoding strand of the Ser 510 bp A l u l DNA fragment containing the tRNA^^ gene as determined by Cribbs (130) (Shizu Hayashi, personal communication). 60 1 2 3 4 61 Figure 7. DNA sequence analysis of pDtA16-2#3: A. Forward d i r e c t i o n . The DNA sequence of the subcloned A l u l DNA fragment was determined by the method of V i e r a and Messing (131) as modified in the Materials and Methods. Lanes 1-4 represent the forward sequencing reactions s p e c i f i c for A, C, G and T re s p e c t i v e l y . The DNA sequence i s shown on the side of the autoradiograph. The sequence represents the coding strand of the 510 bp A l u l DNA fragment containing the tRNA^^ gene. This sequence has not been previously documented. 62 6 3 Figure 8. DNA sequence analysis of pDtA16-2#3: B. Reverse d i r e c t i o n . The DNA sequence of the subcloned A l u l DNA fragment was determined by the method of Vi e r a and Messing (131) as modified in the Materials and Methods. Lanes 1-4 represent the reverse sequencing reactions s p e c i f i c for A, C, G and T res p e c t i v e l y . The DNA sequence i s shown on the side of the autoradiograph. The sequence i s i d e n t i c a l to the noncoding strand of Ser the 510 bp A l u l DNA fragment containing the tRNA^^ gene as determined by Cribbs (130). 64 65 Figure 9. DNA sequence analysis of pDtH16#l: A. Forward d i r e c t i o n . The DNA sequence of th subcloned Hhal DNA fragment was determined by the method of V i e r a and Messing (131) as modified in the Materials and Methods. Lanes 1-4 represent the forward sequencing reactions s p e c i f i c for A, C, G and T r e s p e c t i v e l y . The DNA sequence i s shown on the side of the autoradiograph. X denotes unspecified nucleotides. The sequence represents the noncoding strand of the 980 bp Hhal DNA fragment containing Ser the tRNA^^^ gene. This sequence has not been previously documented. 67 Figure 10. DNA sequence analysis of pDtH16#l: B. Reverse d i r e c t i o n . The DNA sequence of the subcloned Hhal DNA fragment was determined by the method of V i e r a and Messing (131) as modified in the Materials and Methods. Lanes 1-4 represent the reverse sequencing reactions s p e c i f i c for A, C, G and T r e s p e c t i v e l y . The DNA sequence i s shown on the side of the autoradiograph. The sequence i s i d e n t i c a l to the coding strand of the Ser 980 bp Hhal DNA fragment containing the tRNA^^ gene as determined by Cribbs (130). 68 69 Figure 11. DNA sequence analysis of pDtH16#5: A. Forward d i r e c t i o n . The DNA sequence of the subcloned Hhal DNA fragment was determined by the method of V i e r a and Messing (131) as modified in the Materials and Methods. Lanes 1-4 represent the forward sequencing reactions s p e c i f i c for A, C, 6 and T r e s p e c t i v e l y . The DNA sequence i s shown on the side of the autoradiograph. The sequence represents the noncoding strand of the Ser 980 bp Hhal DNA fragment containing the tRNA^^ gene. This sequence has not been previously documented. 70 71 Figure 12. DNA sequence analysis of pDtH16#5: B. Reverse d i r e c t i o n . The DNA sequence of the subcloned Hhal DNA fragment was determined by the method of Viera and Messing (131) as modified in the Materials and Methods. Lanes 1-4 represent the forward sequencing reactions s p e c i f i c for A, C, G and T r e s p e c t i v e l y . The DNA sequence i s shown on the side of the autoradiograph. The sequence i s i d e n t i c a l the coding strand of the Ser 980 bp Hhal DNA fragment containing the tRNA^^ gene as determined by Cribbs (130). 72 73 sequences of the other 3 plasmids in the reverse d i r e c t i o n (Figure 8, 10, 12) were i d e n t i c a l to the DNA sequence of pDtl6 described by Cribbs (130). The o r i e n t a t i o n of the 510 bp A l u l DNA i n s e r t containing the Ser tRNA ? 7 7 gene in pUC8 was exactly as predicted by the r e s t r i c t i o n enzyme analysis (Figure 4a). The 980 bp Hhal DNA i n s e r t in pDtH16#5 was oriented in pUC8 in a d i r e c t i o n opposite to that of pDtA16-2#l. The DNA inserts in pDtA16-2#3 and pDtH16#l were oriented in the same d i r e c t i o n in pUC8 as described in Figure 4c. C. Construction of pDt73x27 Set* The plasmid pDt73 contains a tRNA^^ gene which lacks an o l i g o dT termination s i t e f o r t r a n s c r i p t i o n (130). The lack of a terminator sequence may be the r e s u l t of a cloning a r t i f a c t . The r e s t r i c t i o n enzyme H i n d l l l was used to generate a genomic l i b r a r y of Drosophila melanogaster cloned DNA from which a series of tRNAs were i s o l a t e d (129). Since there Ser is a H i n d l l l s i t e 6 bp 3' to the tRNA^,^ gene then i t i s quite Ser possible that the termination s i t e for the tRNA.,, was removed 474 during cloning. To introduce a termination sequence into pDt73 without a f f e c t i n g the i n t e g r i t y of the gene, the following strategy was applied. Ser The plasmid pDt27 contains a 6.4 kb i n s e r t harboring two tRNA^^ Arc* Ser genes and four tRNA genes (139). The structure of the tRNA^^ Ser gene d i f f e r s from the tRNA^^ gene only in the A -> C point mutation at p o s i t i o n 34 of the coding region. A unique Mspl s i t e located Ser 74 bp into the s t r u c t u r a l element of both tRNA genes could serve as a Ser convenient s i t e f o r i n s e r t i n g the terminator sequence from tRNA^^ 74 Figure 13. The strategy used to reconstruct a tRNA^,^ gene containing a termination sequence. Ser To construct a tRNA^^ gene with a t r a n s c r i p t i o n termination s i t e a 327 bp Sau96I-MspI DNA fragment from pDt73 (a.) was l i g a t e d to a 490 bp Mspl-Ddel DNA fragment from pDt27 (b.) p r i o r to i n s e r t i o n into the Smal s i t e of pUC8. The r e s u l t i n g plasmid pDt73X27 (c.) would contain a Sen tRNA^^ gene with a stretch of 6 T residues 16 bp from the 3' end of the gene which would serve as a termination s i g n a l for RNA polymerase Sec I I I . The tRNA genes are shown by open boxes. The f i l l e d boxes depict pBR322 DNA sequences. Drosophila DNA sequences flanking the genes are shown as s o l i d l i n e s . Cleavage s i t e s f o r the r e s t r i c t i o n endonucleases Sau96I, Hspl, H i n d l l l and Ddel are indicated. 75 CO 0) Q 0) X) o CO 0) 3 (0 CO •D_— .5 a x - 1 (O O) fl> CO Q Q. <N S a x Q a a . 2 a 5 a </)" 2 co O) 3 CO a o o TJ . c 76 Ser gene into the tRNA^^ gene without a f f e c t i n g the i n t e g r i t y of the Ser tRNA^ 7 4 gene (Figure 13). P a r t i a l r e s t r i c t i o n endonuclease maps for the plasmids pDt73 and pDt27 (shown in Figure 13) were constructed from t h e i r DNA sequences (130, 139). From the r e s t r i c t i o n endonuclease maps a 517 bp Sau 961 fragment Ser derived from pDt73 would contain the tRNA^^ gene and a single Mspl s i t e . Digestion of t h i s fragment with Mspl would y i e l d a 327 bp Sau 961-Mspl DNA fragment containing the 5' ha l f of the tRNA gene. Double digestion of pDt27 with Ddel and H i n d l l l would separate the two Ser ArK tRNA 4 4 4 genes from the four tRNA 6 genes on a DNA fragment of approximately 1020 bp in length. The 1020 bp D d e l - H i n d l l l DNA fragment when digested with Mspl would generate 4 DNA fragments, of which a 490 bp Ser Mspl-Ddel DNA fragment would contain the 3* h a l f of a tRNA,,, gene 444 along with the termination s i t e f o r.that gene. L i g a t i n g the 327 bp Sau 961-MspI DNA fragment from pDt73 to the 490 bp Mspl-Ddel DNA fragment Ser from pDt27 would r e s u l t in a tRNA^^ gene with a stre t c h of 6 T's 16 bp from the 3' end of tRNA gene which would serve as a termination s i t e f or t r a n s c r i p t i o n . Fragments generated from the digestion of 3 yg of pDt73 with 5 units of Sau 961 and 3 yg of pDt27 double digested with 5 units of Ddel and 4 units of H i n d l l l at 37°C for 4 hours were electrophoresed on a 1.6% agarose gel containing ethidium bromide. A f t e r the gel was photographed, DNA in the gel was denatured, and then tr a n s f e r r e d onto a n i t r o c e l l u l o s e f i l t e r as in the Methods. As shown in Figure 14, the 517 bp Sau 961 DNA fragment from pDt73 and the 1020 bp D d e l - H i n d l l l DNA 77 Figure 14. I d e n t i f i c a t i o n of DNA fragments derived from pDt27 and pDt73 Ser which contain tRNA genes. Fragments generated from the digestion of pDt27 with Ddel and H i n d l l l (lane 2) and pDt73 with Sau96I (lane 3) were subjected to 1.6% agarose gel electrophoresis f or 4 hours at 120 V (10 V/cm). The gel was stained with ethidium bromide and photographed under UV l i g h t . The DNA fragments were transferred to a n i t r o c e l l u l o s e f i l t e r by the method of Southern (128), Ser hydridized with 3'-end l a b e l l e d tRNA ^ and autoradiographed. A 1033 bp Dde-Hindlll fragment derived from pDt27 (lane 4) and a 517 bp Sau96I fragment from pDt73 (lane 5) hybridized with the probe. Arrows show corresponding DNA bands in lane 2 and 3. Lane 1 shows pBR322 DNA cleaved with Hspl as molecular weight marker (143). 78 79 fragment from pDt27 were found to contain tRNA genes by h y b r i d i z a t i o n Ser* with a tRNA 7 7 7 probe. To i s o l a t e large amounts of the two DNA fragments (517 bp Sau 961 and the 1020 bp D d e l - H i n d l l l DNA fragments), 20 yg of pDt73 were treated with 25 units of Sau 961 for 6 hours at o 37 C. To i s o l a t e the other r e q u i s i t e fragment 20 yg of pDt27 was digested with 25 units of Ddel and 10 units of H i n d l l l f o r 6 hours at o 37 C. The DNA was electrophoresed on a 1.6% agarose gel containing ethidium bromide (4 yg of DNA/well). The 517 bp Sau 961 and the 1020 bp D d e l - H i n d l l l DNA fragments were el e c t r o e l u t e d into a trough cut out of the gel j u s t ahead of each DNA band. Af t e r e l e c t r o e l u t i o n , the ethidium bromide was removed from the DNA by 3 extractions with n-butanol followed by 1 extraction with water-saturated ether. The DNA was p r e c i p i t a t e d twice with 95% ethanol. The p u r i f i e d DNA fragments were resuspended in 20 y l of Mspl buffer containing 12 units of Mspl. The reactions were incubated at 37°C for 4 hours p r i o r to electrophoresis i n a 6% polyacrylamide g e l . The 327 bp Sau 96 I-Mspl DNA fragment from pDt73 and the 490 bp Mspl-Ddel DNA fragment were i d e n t i f i e d by fluorescence of the gel a f t e r ethidium bromide st a i n i n g . Gel s l i c e s containing the 327 bp and the 490 bp DNA fragments were cut out of the polyacrylamide g e l . The DNA was e l e c t r o e l u t e d f o r 5 hours and p u r i f i e d as described in the Materials and Methods (section G). Since l i g a t i o n reactions containing the 327 bp Sau 96 I-Mspl and 490 bp Mspl-Ddel DNA fragments would r e s u l t i n 7 possible DNA inserts of 5 d i f f e r e n t s i z e s , the conditions of the reaction were set to l i m i t the Sen degree of s e l f l i g a t i o n to the 3' portion of the tRNA gene. 80 Reaction mixtures of 10 y l volumes containing approximately a 3 to 1 molar r a t i o of the 327 bp DNA fragment to the 490 bp DNA fragment, 2 units of DNA l i g a s e the DNA li g a s e buffer were incubated at 4°C overnight to maximize the annealing of the 2 bp 5* extended ends r e s u l t i n g from Mspl digestion of DNA. The l i g a t i o n reactions were terminated by heating to 65°C f o r 10 min. The 5' extended ends of the l i g a t e d DNA fragments (the Sau 961, the Ddel and the Mspl generated ends) were f i l l e d i n by adding each of the 4 dNTPs to a f i n a l concentration of 0.3 mM and 2 units of Klenow polymerase and incubating f o r 30 min at 25°C. The r e s u l t i n g blunt ended DNA fragments were inserted into the Smal s i t e of pUC8 as previously described f o r the subcloning of pDtl6 (section B of the Results). E. c o l i (JM 83) was transformed with the l i g a t e d DNA and the bact e r i a containing recombinant plasmids were screened f o r as described in the Materials and Methods (sections Q and R). Small scale plasmid preparations (133) were made from 72 b a c t e r i a l colonies which were selected by t h e i r resistance to a m p i c i l l i n and t h e i r i n a b i l i t y to produce colour on an indicato r plate. The plasmid DNAs were electrophoresed on a 0 .77o agarose gel to determine t h e i r size with respect to pUC8 as a con t r o l . The 35 plasmid DNAs which were found to be larger than pUC8 were treated with 3 units of each, H i n d l l l and EcoRI, i n 20 y l of H i n d l l l o buffer f o r 3 hours at 37 C to l i b e r a t e cloned i n s e r t s . The sizes of the DNA inserts were analyzed by 1.0% agarose gel electrophoresis. Figure 15 represents 11 of the 35 plasmids found to contain i n s e r t s . The gel shows 5 classes of DNA fragments r e s u l t i n g from digesting recombinant plasmids with H i n d l l l and EcoRI. The sizes of the classes of DNA fragments are as 81 predicted by the possible l i g a t i o n reactions. One class of DNA fragment containing the 880 bp insert corresponded to the l i g a t i o n product of a 327 bp Sau 96 I-Mspl DNA fragment and a 490 bp Ddel-Mspl DNA fragment along with approximately 40 bp of pUC8 DNA. Four of the nine plasmids shown to contain t h i s i n s e r t were treated with Mspl, EcoRI and H i n d l l l to determine 1) whether Mspl cleaves the in s e r t and 2) to orientate the in s e r t with respect to the H i n d l l l and EcoRI s i t e s in the vector. Reactions containing 15 y l aliquots of the 4 plasmid DNAs (3 yg), 5 units of each r e s t r i c t i o n endonuclease, Mspl, H i n d l l l and EcoRI and the appropriate buffer were incubated for 3 hours at 37°C. The r e s u l t i n g DNA fragments were electropheresed on a 7% polyacrylamide g e l . The gel was stained with 0.1% ethidium bromide and photographed under UV l i g h t . Figure 16 shows that two plasmids pDt73x27 #54 and #57 generate i d e n t i c a l r e s t r i c t i o n fragments patterns. The other two plasmids pDt73x27#44 and #62 also show i d e n t i c a l r e s t r i c t i o n endonuclease fragment patterns i n t h i s case f o r the same i n s e r t , but in opposite o r i e n t a t i o n . To confirm the construction of the plasmids and the ori e n t a t i o n of the 880 bp i n s e r t i o n pUC8, dideoxy chain terminator DNA sequencing was performed on both pDt73x27#44 and pdt73x27#54. The plasmids pDt73x27#44 and #54 were prepared as in section A of the Methods. The double CsCl density gradient p u r i f i e d plasmid DNA (20 yg) was treated with 30 yg/ml of DNase free RNase for 1 hour at 37°C in 50 y l of H i n d l l l buffer to degrade contaminating RNA. The DNA was d i l u t e d to 300 y l before a p p l i c a t i o n to a 1 M NaCl gradient (section T of the Methods). A f t e r 6 hours of centrifugation the DNA was p r e c i p i t a t e d twice 82 Figure 15. I d e n t i f i c a t i o n of pDt73X27 hybrid plasmids. Recombinant plasmid DNAs found to be larger than pUC8 were digested with EcoRI and H i n d l l l to l i b e r a t e the cloned i n s e r t . The DNA fragments were analyzed by 1.0% agarose gel electrophoresis. The gel was stained with 0.1% ethidium bromide and photographed. Lanes 1-11 are a sample of the 35 plasmids containing i n s e r t s . There are 5 classes of cloned fragments (1030 bp, 880 bp, 710 bp, 530 bp, and 380 bp) as predicted by the possible l i g a t i o n reactions. Lane 12 shows pDt27 DNA digested with H i n d l l l and Ddel (139) and lane 13 shows pBR322 DNA cleaved with A l u l (143). Both lane 12 and lane 13 serve as molecular weight markers. 83 1 2 3 4 5 6 7 8 9 10 11 12 13 1.1.......... 880 bp 530 bp fe 1030 bp < 710 bp * 380 bp 84 Figure 16. Determination of pDt73X27 hybrid plasmid organization. Four hybrid plasmids containing an 880 bp i n s e r t were cleaved with Mspl, EcoRI and H i n d l l l and about 2 yg of DNA from each, pDt73X27#44 (land 2), #54 (lane 3), #57 (lane 4) and #62 (lane 5) were electrophoresed on a 7% polyacrylamide gel at 140V for 4 hours (10 V/cm). The gel was stained with ethidium bromide and was photographed under UV l i g h t . pUC8 DNA digested wih Mspl, Hind I I I and EcoRI was also electrophoresed (lane 1) to provide size markers. Arrows to the r i g h t of the gel show the 490 bp Ddel-Mspl fragment derived from pDt27 while the arrows to the l e f t of the gel show the 327 bp Sau96I-MspI fragment o r i g i n a t i n g from pDt73. 85 1 2 3 4 5 86 with 95% ethanol, resuspended i n 40 y l of TE buffer, and then s p l i t into two, 20 y l al i q u o t s . The DNA subjected to the forward dideoxy sequencing reaction was f i r s t digested with 6 units of EcoRI while DNA subjected to the reverse reaction was digested with 6 units of H i n d l l l to completion at 37°C. The l i n e a r i z e d DNA was electrophoresed i n a 0.77. agarose gel to assess the l e v e l of digestion and to quantify the amount of DNA. The dideoxy sequencing reactions were c a r r i e d out as described (section U of the Methods). The DNA sequences of pDt73x27#54 in the forward d i r e c t i o n (Figure 17A) and of pDt73x27#44 in the reverse d i r e c t i o n (Figure 17B) were found to be i d e n t i c a l to the 490 bp DNA fragment from pDt27 from the Ddel s i t e towards the Mspl s i t e in the tRNA gene. The DNA sequences of pDt73x27#54 in the reverse d i r e c t i o n (Figure 17D) and of pDt73x27#44 in the forward d i r e c t i o n (Figure 17C) corresponds to the DNA sequence of the 327 bp DNA fragment from pDt73, from the Sau 961 s i t e towards the Mspl s i t e . The DNA sequence of the ends of the two plasmids shows that the newly constructed Sec tRNA^^ gene was cloned into pUC8 in both o r i e n t a t i o n and that the sequenced portion of the constructed gene contained no alt e r e d bases. D. Construction of pDtBH27 Ser A DNA sequence analysis of the two tRNA^^ genes and flanking sequences contained in pDt27 revealed that there were no convenient r e s t r i c t i o n endonuclease s i t e s which would be useful to separate the two genes (139). However, a 1230 bp BamHI-Hindlll DNA fragment would i s o l a t e d 87 Figure 17. DNA sequence analysis of pDt73X27#44 and #54. Plasmid DNA subjected to forward dideoxy DNA sequence reactions were digested with EcoRI (A. and C.) while plasmid DNA subjected to reverse reactions were cleaved with H i n d l l l (B. and D.). A. and D. show the DNA sequence of pDt73X27#54. B. and C. show the DNA sequence of pDt73X27#44. Lanes 1-4 represent the sequencing reactions s p e c i f i c f o r A, C, G and T respectively. The DNA sequence shown on the side of the autoradiographs A. and B. are i d e n t i c a l to a segment of the coding strand of the 490 bp Ddel-Mspl fragment derived from pDt27 as determined by Newton (139). The DNA sequence shown on the side of the autoradiographs C. and D. are i d e n t i c a l to a segment of the noncoding strand of the 327 bp Sau 961-MspI fragment derived from pDt73 as determined by Cribbs (130). 88 A. 1 2 3 4 B. 5 6 7 8 89 90 Ser Arg the tRNA^^^ genes from the tRNA & genes also contained in pDt27 and could e a s i l y be subcloned into the cloning vector pUC8. The DNA fragments generated from the digestion of 4 yg of the plasmid pDt27 with 4 units of H i n d l l l and 4 units of BamHI in a 20 y l reaction volume containing BamHI buffer for 3 hours at 37°C were electrophoresed in a 1.4% agarose gel containing ethidium bromide. The gel was monitored by UV fluorescence of the stained DNA and was photographed (Figure 18). The DNA in the gel was transferred onto n i t r o c e l l u l o s e f i l t e r s , and then hybridized with 3* end l a b e l l e d Ser tRNA^ 7 7 as described in the Materials and Methods. As predicted a 1230 bp BamHI-Hindlll DNA fragment was found to hybridize the l a b e l l e d Ser tRNA 7 probe. In a reaction volume of 50 y l , 20 yg of pDt27 was digested with 20 units of each, BamHI and H i n d l l l , under conditions already described. The r e s u l t i n g DNA fragments were separated by 1.4% preparative agarose gel electrophoresis. The 1230 bp DNA fragment Ser containing the two tRNA^^ genes was eluted by electrophoresis of the fragment into a trough f i l l e d with buffer (section H of the Materials and Methods). The ethidium bromide interchelated in the DNA was removed by 3 extractions with n-butanol followed by 1 extraction with water-saturated ether. The BamHI-Hindlll DNA fragment was p r e c i p i t a t e d twice with 95% ethanol, dried i n vacuo and then resuspended in 20 y l of TE buffer. To approximate the amount of DNA recovered by e l e c t r o e l u t i o n , the fluorescence of a 3 y l aliquot of the i s o l a t e d DNA fragment electrophresed on a 1.4% agarose gel containing ethidium bromide was compared to the fluorescence of 1 yg of BamHI, H i n d l l l digested pDt27 91 Figure 18. I d e n t i f i c a t i o n of a Hindlll-BamHI fragment from pDt27 containing tRNA... genes. 444 pDt27 DNA (3 yg) was digested with BamHI and H i n d l l l , electrophoresed i n a 1.4% agarose gel at 120V f o r 4 hours. The gel was stained with ethidium bromide and photographed under UV l i g h t (lane 1). The DNA in the gel was transferred to n i t r o c e l l u l o s e f i l t e r s and Ser hybridized to the 3' end l a b e l l e d tRNA^ probe. Only a 1230 bp DNA Ser fragment hybridized the tRNA probe (lane 2). This DNA fragment corresponds in size to the predicted BamHI-Hindlll fragment derived from pDt27. 92 93 electrophoresed on the same gel. The remaining 17 y l of DNA was stored at -20°C. The plasmid pUC8 (10 yg) was l i n e a r i z e d f o r the subcloning of the 1230 bp DNA fragment derived from pDt27 by digestion with the r e s t r i c t i o n endonuclease, BamHI and H i n d l l l , as described i n the Materials and Methods. The 1230 bp DNA fragment was inserted into the BamHI and H i n d l l l s i t e s of pUC8 in 10 y l l i g a t i o n reactions containing 1, 2 or 3 f o l d molar excess of the DNA fragment over pUC8 DNA and 0.3 yg of l i n e a r i z e d pUC8. Linea r i z e d pUC8 was tested f o r i t s a b i l i t y to serve as substrate i n the absence of in s e r t DNA. The conditions f o r the l i g a t i o n reactions were the same as those described in section P of the Methods. Transformation of the l i g a t e d DNA into E. c o l i JM 83 and the se l e c t i o n of the recombinant plasmids were as outlined i n section Q and section R of the Methods resp e c t i v e l y . Plasmid DNAs i s o l a t e d from 9 out of 12 p o s i t i v e clones by the method of Holmes and Quigley (133, section S of the Methods) were shown to contain the 1230 bp ins e r t when 15 y l of plasmid DNA was cleaved with 3 units of BamHI and 3 units of H i n d l l l under the appropriate conditions and subjected to 1.1% agarose gel electrophoresis. One new subclone, r e f e r r e d to as pDtBH27#l, was p u r i f i e d from a one 1 b a c t e r i a l culture grown in M9 s a l t s (section A of the Methods) and was prepared f o r chain terminator DNA sequencing as described above except that the DNA used as template f o r the forward sequencing reaction was digested to completion with 6 units of BamHI at 37°C. The dideoxy DNA sequencing 94 Figure 19. DNA sequence analysis of pDtBH27#l. pDtBH27#l DNA was cleaved with EcoRI f o r use as template in the forward dideoxy DNA sequencing reaction (A) and with H i n d l l l f o r use as template in the reverse dideoxy DNA sequencing reaction (B). Lanes 1-4 represent sequencing reactions s p e c i f i c f o r A, C, G and T r e s p e c t i v e l y . The DNA sequence shown on the side of autoradiograph A. i s i d e n t i c a l to the noncoding strand of the 1230 bp fragment from the H i n d l l l s i t e into the i n s e r t . Autoradiograph B. shows the DNA sequence of a segment of the coding strand of the 1230 bp fragment from the BamHI s i t e into the i n s e r t (139) . 95 96 reactions were performed as described in the Materials and Methods (section T). As shown in Figure 19 the segment of DNA sequence of both the forward and the reverse reactions correspond exactly to the sequence of the BamHI-Hindlll i n s e r t o r i g i n a t i n g from pDt27 as described by Newton (139). Sec* This DNA fragment contains the two tRNA^^ genes. I I . Properties of Drosophila (Schneider II) c e l l - f r e e extracts Ser A. T r a n s c r i p t i o n products derived from tRNA genes The supernatant f r a c t i o n r e s u l t i n g from high speed c e n t r i f u g a t i o n (100,000 x g) of Drosophila (Schneider II) culture c e l l extracts was used to support homologous in. v i t r o t r a n s c r i p t i o n reactions which accurately Sec* and s e l e c t i v e l y transcribe tRNA genes. Figure 20 shows that Seir t r a n s c r i p t i o n of the d i f f e r e n t tRNA genes resulted in the synthesis of a d i s c r e t e set of RNAs c h a r a c t e r i s t i c of each gene. A f t e r 90 min. of incubation at 23°C the tRNA t r a n s c r i p t s were synthesized as a major RNA species 20-25 nucleotides longer than the mature form of the tRNA together with other minor bands. The weaker bands represent RNAs which are heterogenous in conformation, terminal sequences, si z e or perhaps nucleotide modifications. Addition of pBR322 to the t r a n s c r i p t i o n reactions (lane 2) in the absence of genes s p e c i f i c templates yielded no d i s c r e t e RNA species. There i s also l i t t l e endogenous a c t i v i t y except for an RNA species which i s perhaps 5S RNA (lane 1). Sen A f t e r electrophoresis, RNA synthesized from the tRNA^^ genes on the plasmids pDt5 (lane 3), pDtl7R (lane 4) and the pDtl6 subclones 97 Figure 20. Tra n s c r i p t i o n of d i f f e r e n t plasmids carrying Drosophila Ser tRNA genes. Tra n s c r i p t i o n reactions containing 0.6 yg of supercoiled pDt5 DNA (lane 3), pDtl7R DNA (lane 4), pDtl6 DNA (lane 5), pDtA16-2#3 DNA (lane 6), pDtH16#l DNA (lane 7), pDtA16-2#l DNA (lane 8), pDtH16#5 DNA (lane 9), pDt27 DNA (lane 10), pDtBH27#l DNA (lane 11), pDt73 DNA (lane 12) and pDt73X27 DNA (lane 13) were performed and analyzed as described i n the Methods. S-100 extract was monitored for endogenous t r a n s c r i p t i o n a c t i v i t y (lane 1). Non s p e c i f i c t r a n s c r i p t i o n from 0.6 yg of supercoiled pBR322 DNA was also monitored (lane 2). Lane 14 shows 3' end Ser l a b e l l e d tRNA^ as size marker. The approximate size of both precursor and mature tRNA species are summarized in Table I I . Table II Ser Summary of the size of the tRNA gene t r a n s c r i p t i o n products Plasmid PDt5 pDtl7R pDtl6 pDtA16-2#3 pDtH16#l pDtA16-2#l pDtH16#5 pDt27 PDtBH27#l pDt73 pDt73X27 Template • Type of t R N A 5 " gene 777 777 777 774 774 774 777 777 444 444 * 444 444 474 4 74 Approximate s i z e of precursor tRNA (nucleotides) 107 101 116 106 100 115 109 107 106 102 101 93 109 107 101 93 115 109 107 101 93 106 102 106 102 108 98 96 117 108 98 117 110 Approximate 85 85 85 85 85 85 85 85 85 s i z e of 74 mature product (nucleotides) * a l s o contains 4 tRNA 8 genes 100 pDtA16-2#l (lane 8) and pDtH16#5 (lane 9) resulted in almost i d e n t i c a l banding patterns suggesting that each template shares not only i d e n t i c a l coding sequences but also very s i m i l a r t r a n s c r i p t i o n s t a r t s i t e s and termination s i t e s (see Table II for the si z e of the i n d i v i d u a l t r a n s c r i p t s ) . T r a n s c r i p t i o n of the subclone pDtH16#l which a Ser tRNA^^ gene o r i g i n a t i n g from the plasmid pDtl6 resulted in a s p e c i f i c set of RNA t r a n s c r i p t s 115, 109, 107, 101, 93 and 85 nucleotides in length (lane 7). A deletion mutant of pDtH16#l re f e r r e d to as pDtA16-2#3 continued to support t r a n s c r i p t i o n (lane 6) even though i t contained only 36 bp of the o r i g i n a l 5* flanking sequence. The products of pDtA16-2#3 t r a n s c r i p t i o n were i d e n t i c a l to those of pDtH16#l t r a n s c r i p t i o n except that the 115 nucleotide t r a n s c r i p t was not apparent. I t appears that 5' flanking sequences removed by the d e l e t i o n were important in the i n i t i a t i o n of the 115 nucleotide RNA, although removal of such sequences does not a f f e c t the synthesis of the other RNA. The t r a n s c r i p t i o n products derived from the plasmid pDtl6 which harbors both a Ser Ser tRNA,,, gene and a tRNA,,, gene were i d e n t i c a l to the RNAs 774 777 Ser synthesized from the subclones pDtH16#l (tRNA^^ gene) and pDtH16#5 ( t R N A ^ gene) (lane 5). The plasmid pDt73 when used as template d i d not support s p e c i f i c RNA synthesis (lane 12). Presumably the i n a b i l i t y of pDt73 to serve as template was due to the lack of an o l i g o T sequence or other 3' sequences (73-75) which have been implicated to be involved in the termination of t r a n s c r i p t i o n and are normally found 3' to Class III genes. Ser Reconstruction of the tRNA gene (pDt73x27) such that the 101 Ser termination sequence from a tRNA^^ gene was fused to the 3' end of SER the tRNA^^, gene resulted in the synthesis of two s p e c i f i c t r a n s c r i p t s , 117 and 110 nucleotides in length (lane 13). The plasmid pDt27 contains 4 tandem repeats of a tRNA A r^ gene together with 2 Ser tRNA^^ genes. A comparison of the t r a n s c r i p t i o n products derived from pDt27 (lane 10) and pDtBH27#l (lane 11), a subclone of pDt27 which Ser contains only the 2 tRNA^^, genes, revealed that the major 108 nucleotide t r a n s c r i p t and the two minor t r a n s c r i p t s 98 and 85 nucleotides Ser in length were synthesized from the tRNA^^ genes whereas the major 98 nucleotide t r a n s c r i p t and the 96 and 69 nucleotide RNAs were ArK synthesized from the tRNA genes. The plasmid pDtBH27#l d i r e c t e d the synthesis of an a d d i t i o n a l t r a n s c r i p t , 117 nucleotides in length. The smallest of the s p e c i f i c RNA products, an 85 nucleotide RNA was constant for a l l DNA templates which supported t r a n s c r i p t i o n . The 85 nucleotide Ser RNA was s i m i l a r in size to the tRNA ? 7 7 marker (lane 14) and may represent the mature tRNA product. The 74 nucleotide t r a n s c r i p t derived Arc from the tRNA genes in pDt27 may also represent the mature form of the tRNA since the tRNA i s approximately that size (139). B. Authenticity of RNA t r a n s c r i p t s d i r e c t e d by pDt5 To demonstrate that the proposed precursor and mature tRNA species were s p e c i f i c t r a n s c r i p t s derived from the tRNA gene on pDt5, l a b e l l e d in v i t r o t r a n s c r i p t i o n products were eluted from bands on polyacrylamide gels and hybridized to pDtl7R DNA bound to n i t r o c e l l u l o s e f i l t e r s . Both pDt5 Ser and pDtl7R DNA contain Drosophila tRNA genes (129). Sequences 102 Figure 21. Authenticity of the tRNA gene t r a n s c r i p t i o n products. The pDtl7R DNA (2 yg) was cleaved with 4 units of H i n d l l l f o r 3 o hours at 37 C. The DNA was electrophoresed in a 0.8% agarose g e l , stained with ethidium bromide and photographed under UV l i g h t (lane 1). The DNA in the gel was transferred to n i t r o c e l l u l o s e f i l t e r s as described 32 by Southern (128). [a- P]UTP l a b e l l e d t r a n s c r i p t i o n products r e s u l t i n g from incubating 0.8 yg of pDt5 DNA in Drosophila Schneider II c e l l extract as described in the Methods were electrophoresed on a denaturing 10% polyacylamide gel and autoradiographed overnight. Both Ser* precursor and mature tRNA^ were excised from the gel and eluted by incubating the gel s l i c e s in 500 y l of solution containing 0.5 M o ammonium acetate and 0.001 M EDTA overnight at 65 C. The eluate was p r e c i p i t a t e d with ethanol and resuspended in 100 y l of TE buffer. The pDt5 t r a n s c r i p t i o n products were hybridized to pDtl7R DNA bound to n i t r o c e l l u l o s e f i l t e r s as described in the Methods. Lane 2 shows an autoradiograph of the f i l t e r a f t e r overnight exposure. The 3350 bp fragment corresponding to pDtl7R i n s e r t DNA hybridized the tRNA species synthesized from pDt5. 103 3350 b p 104 flanking these genes are t o t a l l y heterologous (130, 139). Thus, i f pDt5 DNA supports t r a n s c r i p t i o n of authentic tRNA species, they should Ser hybridize to that part of pDtl7R DNA corresponding to the tRNA gene. The r e s u l t s of such a h y b r i d i z a t i o n are shown in Figure 21. Only the 3350 bp Drosophila insert hybridized with the iri v i t r o t r a n s c r i p t i o n products thus demonstrating the a u t h e n t i c i t y of the tRNA species. C. E f f e c t of DNA concentration on the rate of t r a n s c r i p t i o n . To examine the t r a n s c r i p t i o n e f f i c i e n c y of the c o l l e c t i o n of Ser tRNA genes, the rate of t r a n s c r i p t i o n was measured as a function of DNA input. T y p i c a l examples of the data obtained for two templates, pDt5 and pDtH16#5 are shown in Figure 22. Figure 22C represents an autoradiograph of the t r a n s c r i p t i o n products r e s u l t i n g from reactions containing these tempates. The positions of the primary t r a n s c r i p t i o n product (RNA-I) and the processed tRNA sized species (RNA-II) are indicated. Quantitative analysis of the RNA synthesized from either template revealed a sigmoidal dependence of t r a n s c r i p t i o n rate on DNA input. The maximum rate of t r a n s c r i p t i o n f o r these two DNA templates occurred at between 0.6 ug-1.0 yg of DNA per 50 y l reaction. At higher l e v e l s of DNA input the t r a n s c r i p t i o n rate decreased markedly but the k i n e t i c s of decrease were not the same for the two templates. These Ser r e s u l t s were c h a r a c t e r i s t i c of a l l tRNA genes tested. The sigmoidal nature of the i n i t i a l portion of the DNA concentration curve made i t d i f f i c u l t to accurately determine an i n i t i a l rate of tRNA synthesis. 105 Figure 22. E f f e c t of DNA concentration on the rate of t r a n s c r i p t i o n of pDt5 and pDtH16#5. The tRNA precursors synthesized a f t e r 90 min in the presence of 32 [a- P]UTP were separated on a polyacrylamide g e l . The gel was autoradiographed to l o c a l i z e the positions of the RNA products (Figure 22C). The p o s i t i o n of the primary t r a n s c r i p t i o n product (RNA-I) and the processed form (RNA-II) are shown. Lanes 1 to 7 show t r a n s c r i p t i o n products from pDt5 and lanes 8 to 14 show t r a n s c r i p t i o n products from pDtH16#5 when reaction mixtures d i d not contain nontemplate DNA. The Cerenkov r a d i a t i o n i n the t r a n s c r i p t i o n products was determined as described i n Methods and was plotted as a function of concentration of pDt5 (o) and pDtl6H#5 (o: Figure 22A) and as a function of gene equivalence (Figure 22B). Gene equivalences were calculated as yg DNA input divided by plasmid molecular weight and normalized to unit values by 13 multipl y i n g by 10 (Table I I I ) . 106 108 Replotting the t r a n s c r i p t i o n rate in Figure 22A as a function of the number of genes in the reaction rather than the amount of DNA, r e s u l t e d in curves which maintained t h e i r sigmoidal shape; however, the maximum rate of t r a n s c r i p t i o n f o r the two templates no longer occurred at the same input (Figure 22B). A f t e r conversion of the amount of DNA to the number of gene equivalences, the input y i e l d i n g the maximum v e l o c i t y of t r a n s c r i p t i o n appeared to s h i f t inversely with respect to the molecular weight of the plasmid containing the gene. These re s u l t s suggested that t r a n s c r i p t i o n was dependent on several f a c t o r s : 1) the amount of DNA added to the reaction mixture; 2) the a b i l i t y of the DNA to act as a template; 3) the molecular weight of the plasmid containing the gene; but, s u r p r i s i n g l y , not the absolute number of genes added to the reaction. Since the rate of t r a n s c r i p t i o n should be d i r e c t l y dependent on gene input, the dependence of the reactions on the amount of DNA rather than the number of genes suggested the presence of some i n h i b i t o r in the reaction could be t i t r a t e d out by higher l e v e l s of DNA. In an attempt to mimic the higher l e v e l s of template which yi e l d e d the maximum rates of t r a n s c r i p t i o n , I added increasing amounts of various types of nontemplate DNA to t r a n s c r i p t i o n reactions containing e i t h e r 0.05 yg or 0.1 yg of the plasmid pDt5 (Figure 23). The r e s u l t s showed that increasing the amount of pBR322 or the synthetic double stranded copolymer poly dGC stimulated the rate of t r a n s c r i p t i o n dramatically. The t r a n s c r i p t i o n of 0.1 yg of pDt5 was stimulated two-fold by pBR322, with the maximum rate r e s u l t i n g from an input of 0.6 to 1.0 yg of DNA. A f i v e - f o l d stimulation was observed when 0.05 yg of pDt5 was used as the 109 Figure 23. Transcription of pDt5 in the presence of nontemplate DNA. Nontemplate DNA was added to the t r a n s c r i p t i o n reactions and the 32 incorporation of [a- P]UTP into t r a n s c r i p t i o n products was followed as described in Methods. The incorporation, measured by Cerenkov r a d i a t i o n , was plotted against the amount of nontemplate DNA added. Tra n s c r i p t i o n reaction contained e i t h e r 0.05 yg of pDt5 as the template DNA and pBR322 as the nontemplate DNA: (o), or 0.1 yg of pDt5 DNA as the template and the following nontemplate DNAs, pBR322 (•); M13mpll (•); poly dAT ( A ) ; poly dGC (A); DNA from B a c i l l u s s u b t i l i s phage (p29 (•). V e l o c i t y ( c p m / h r x l O ^ ) i I < I I I ' l l | , I L I I L 1 1 1 L I l l template. The maximum rate of t r a n s c r i p t i o n was s t i l l achieved at 0.6 to 1.0 yg of pBR322, however, the l e v e l of t r a n s c r i p t i o n at the maximum was lower than that observed with 0.1 yg of pDt5. Thus the rate of t r a n s c r i p t i o n of template DNA in the presence of pBR322 was proportional to the number of genes in the reaction. The amounts of pBR322 and poly dGC required for maximum t r a n s c r i p t i o n rates corresponded to the amount of plasmid required for maximum synthesis in the absence of non-template DNA. Not a l l DNA or DNA analogues were e f f e c t i v e at stimulating t r a n s c r i p t i o n . The addition of increasing amounts of the double stranded copolymer poly dAT or single stranded phage DNA (M13mpll) l e d to a decrease in the rate of synthesis of tRNA precursor (Figure 23). DNA from a B a c i l l u s s u b t i l i s phage, (p29, which i s approximately 62% AT (141) s l i g h t l y stimulated t r a n s c r i p t i o n of 0.1 yg pDt5 when present at l e v e l s up to 0.3 yg. However, increasing amounts of <p29 DNA beyond 0.6 yg resulted in rapid loss of t r a n s c r i p t i o n . F i n a l l y , t r a n s c r i p t i o n of pDt5 was p a r t i c u l a r l y s e n s i t i v e to heparin. Synthesis of tRNA precursors was t o t a l l y i n h i b i t e d at l e v e l s as low as 0.05 yg/50 y l (data not shown). I t i s not c l e a r how the i n h i b i t o r which i s sequestered by high DNA l e v e l s functions to decrease t r a n s c r i p t i o n . Analysis of the template DNA a f t e r incubation under t r a n s c r i p t i o n conditions using agarose gel electrophoresis suggested no nuclease a c t i v i t y was present in the extracts. However, low l e v e l s of nuclease might act at low DNA concentrations and not be detectable by the methods I have t r i e d . 112 D. K i n e t i c Analysis of tRNA^ e r genes Based on the observations described above, the k i n e t i c analysis of Ser t r a n s c r i p t i o n of the tRNA genes was performed with pBR322 DNA added to reaction mixtures to maintain a t o t a l DNA concentration of 1.0 yg per 50 y l reaction. In the presence of a t o t a l DNA concentration of 1.0 yg the rate of t r a n s c r i p t i o n no longer showed sigmoidal dependence on DNA concentration (Figures 24A-30A). The observed rate of t r a n s c r i p t i o n was a hyperbolic function of gene input, reminiscent of steady state enzyme k i n e t i c s . In the absence of nontemplate DNA low inputs of template DNA had shown no tRNA precursor synthesis. However, when nontemplate DNA was added to keep a t o t a l DNA input of 1.0 yg, low DNA inputs yielded a reaction v e l o c i t y nearly proportional to the concentration of template. At higher template concentrations the rate of tRNA precursor synthesis increased less r a p i d l y u n t i l a maximum v e l o c i t y was achieved. For a l l of the cloned genes the maximum rate of tRNA precursor production was the same in the presence and absence of the nontemplate DNA, i n d i c a t i n g no loss of a c t i v i t y due to the addition of the nontemplate DNA. Since these r e s u l t s were consistent with c l a s s i c a l one substrate Ser enzyme k i n e t i c s , I examined the c o l l e c t i o n of tRNA genes and measured the k i n e t i c parameters Km and Vmax using the Hichaelis-Menten equation. Ser The Vmax and Km of the tRNA genes were measured by a double r e c i p r o c a l p l o t analogous to a Lineweaver-Burke pl o t (Figures 24B-30B). The determination of the k i n e t i c parameters appeared to be quite accurate since most of the data points conformed to the expected l i n e a r r e l a t i o n s h i p , except f o r the point corresponding to the lowest DNA 113 Figure 24. Tra n s c r i p t i o n of pDt5 in the presence and absence of pBR322 DNA. Transcription reactions using pDt5 DNA were c a r r i e d out in the absence (4) or the presence of pBR322 DNA added to keep the t o t a l DNA concentration at 1.0 yg per 50 y l reaction (•) an the products were analyzed as described i n Methods. In Figure 24A the v e l o c i t y of the t r a n s c r i p t i o n reaction (cpm/hr) i s plotted as a function of the template concentration (M). Lanes 1 to 6 in the inset show t r a n s c r i p t i o n products derived from pDt5. Figure 24B i s the data obtained from t r a n s c r i p t i o n i n the presence of pBR322 plotted as the r e c i p r o c a l of the v e l o c i t y of the reaction (cpm/hr) ^ versus the r e c i p r o c a l of the concentration of the template DNA (M _ 1). 114 115 15 3 0 45 60 I / [Template] , nM~' 116 Figure 25. Transcription of pDtl7R in the presence and absence of pBR322 DNA. Transcription reactions using pDtl7R DNA were c a r r i e d out in the absence (A) or the presence of pBR322 DNA added to keep the t o t a l DNA concentration at 1.0 yg per 50 y l reaction (•) and the products were analyzed as described i n Methods. In Figure 25A the v e l o c i t y of the t r a n s c r i p t i o n reaction (cpm/hr) i s plotted as a function of the template concentration (M). Lanes 1 to 6 in the inset show t r a n s c r i p t i o n products derived from pDtl7R. Figure 25B i s the data obtained from t r a n s c r i p t i o n in the presence of pBR322 plotted as the r e c i p r o c a l of the v e l o c i t y of the reaction (cpm/hr) versus the r e c i p r o c a l of the concentration of the template DNA ( M - 1 ) . 117 1 1 1 1 1 r 1 2 3 4 5 6 0 .4 0.8 12 [Temp la te ] , n M 119 Figure 26. Transcription of pDtA16-2#l in the presence and absence of pBR322 DNA. Tran s c r i p t i o n reactions using pDtA16-2#l DNA were c a r r i e d out in the absence (A) or the presence of pBR322 DNA added to keep the t o t a l DNA concentration at 1.0 yg per 50 y l reaction (•) and the products were analyzed as described i n Methods. In Figure 26A the v e l o c i t y of the t r a n s c r i p t i o n reaction (cpm/hr) i s plotted as a function of the template concentration (M). Lanes 1 to 6 in the inset show t r a n s c r i p t i o n products derived from pDtA16-2#l. Figure 26B i s the data obtained from t r a n s c r i p t i o n in the presence of pBR322 plotted as the r e c i p r o c a l of the v e l o c i t y of the reaction (cpm/hr) 1 versus the r e c i p r o c a l of the concentration of the template DNA (M "S . 1 2 0 OS 1.6 2 .4 [Temp la te ] , n M 121 122 Figure 27. Transcription of pDtH16#5 in the presence and absence of pBR322 DNA. Tran s c r i p t i o n reactions using pDtH16#5 DNA were c a r r i e d out in the absence (A) or the presence of pBR322 DNA added to keep the t o t a l DNA concentration at 1.0 ug per 50 y l reaction (•) and the products were analyzed as described in Methods. In Figure 27A the v e l o c i t y of the t r a n s c r i p t i o n reaction (cpm/hr) i s plo t t e d as a function of the template concentration (M). Lanes 1 to 6 in the inset show t r a n s c r i p t i o n products derived from pDtH16#5. Figure 27B i s the data obtained from t r a n s c r i p t i o n in the presence of pBR322 plotted as the r e c i p r o c a l of the v e l o c i t y of the reaction (cpm/hr) ^ versus the r e c i p r o c a l of the concentration of the template DNA (M _ 1). 123 1 1 1 1 r 1 2 3 4 5 6 [Template], nM 124 125 Figure 28. Transcription of pDtA16-2#3 in the presence and absence of pBR322 DNA. Tra n s c r i p t i o n reactions using pDtA16-2#3 DNA were c a r r i e d out in the absence (A) or the presence of pBR322 DNA added to keep the t o t a l DNA concentration at 1.0 yg per 50 y l reaction (•) and the products were analyzed as described i n Methods. In Figure 24A the v e l o c i t y of the t r a n s c r i p t i o n t r e a c t i o n (cpm/hr) i s plotted as a function of the template concentration (M). Lanes 1 to 6 in the inset show t r a n s c r i p t i o n products derived from pDtA16-2#3 DNA. Figure 28B i s the data obtained from t r a n s c r i p t i o n in the presence of pBR322 plotted as the r e c i p r o c a l of the v e l o c i t y of the reaction (cpm/hr) versus the r e c i p r o c a l of the concentration of the template DNA (M "S . 126 127 128 Figure 29. Transcription of pDtH16#l in the presence and absence of pBR322 DNA. Tran s c r i p t i o n reactions using pDtH16#l DNA were c a r r i e d out in the absence (A) or the presence of pBR322 DNA added to keep the t o t a l DNA concentration at 1.0 yg per 50 y l reaction (•) and the products were analyzed as described in Methods. In Figure 29A the v e l o c i t y of the t r a n s c r i p t i o n reaction (cpm/hr) i s plotted as a function of the template concentration (M). Lanes 1 to 6 in the inset show t r a n s c r i p t i o n products derived from pDtH16#l DNA. Figure 29B i s the data obtained from t r a n s c r i p t i o n in the presence of pBR322 plotted as the r e c i p r o c a l of the v e l o c i t y of the reaction (cpm/hr) ^ versus the r e c i p r o c a l of the concentration of the template DNA (M ' S . 130 131 Figure 30. Tra n s c r i p t i o n of pDt73X27 in the presence and absence of pBR322 DNA. Tran s c r i p t i o n reactions using pDt73X27 DNA were c a r r i e d out in the absence (A) or the presence of pBR322 DNA added to keep the t o t a l DNA concentration at 1.0 yg per 50 y l reaction (•) and the products were analyzed as described in Methods. In Figure 30A the v e l o c i t y of the t r a n s c r i p t i o n reaction (cpm/hr) i s plotted as a function of the template concentration (M). Lanes 1 to 6 in the inset show t r a n s c r i p t i o n products derived from pDt73X27 DNA. Figure 30B i s the data obtained from t r a n s c r i p t i o n in the presence of pBR322 plo t t e d as the r e c i p r o c a l of the v e l o c i t y of the reaction (cpm/hr) ^ versus the r e c i p r o c a l of the concentration of the template DNA (M 1 ) . 133 134 concentration used. The high degree of l i n e a r i t y substantiated the v a l i d i t y of the use of the Lineweaver-Burke method. The Km determined by t h i s method was treated as a Km apparent due to the complexity of the reaction and w i l l be subsequently termed Kmapp. The Kmapp i s a measure of the t r a n s c r i p t i o n complex a f f i n i t y f o r the DNA when a l l other components of the t r a n s c r i p t i o n reaction are held constant and refer s to the a f f i n i t y of the stable form of the t r a n s c r i p t i o n complex f o r the DNA template (see Discussion). The normalized rate of tRNA precursor synthesis as a function of substrate concentration, was measured using concentrations of template DNA which approximated f i r s t order dependence. The values of Vs demonstrate the stimulation of t r a n s c r i p t i o n by the addition of pBR322. Ser The c o l l e c t i o n of tRNA genes subjected to k i n e t i c analysis Ser Ser included four tRNA ? 7 7 genes and 3 allogenes. The tRNA ? 7 7 genes were i d e n t i c a l within the tRNA coding region, but varied considerably in the 5' and 3' flanking regions. Thus the set of Ser tRNA 7 7 7 genes allowed me to analyze the e f f e c t s of a l t e r a t i o n s in 5' flanking sequence on the k i n e t i c parameters of tRNA synthesis. The 3 allogenes contained two point mutations within the tRNA coding regions and were completely heterologous i n the 5* flanking sequences. The nature of Ser the point mutations and the c o l l e c t e d k i n e t i c parameters f o r the tRNA genes are indicated i n Table I I I . Analysis of the data i n Table I I I allowed the following generalizations. The Kmapp of the t r a n s c r i p t i o n complex was only s l i g h t l y a l t e r e d by changes in the DNA sequence flanking the 5* end of the Ser —10 tRNA 7 7 7 genes. The range of Kmapp was between 1.0 x 10 M (for Table III Kinetic parameters from in v i t r o t r a n s c r i p t i o n of tRNA^ e r genes Plasmid MW of Type of Vs Vs V m a x K m a p p a r e n t + DNA tRNA S e r -pBR +pBR pmoles of CM) (daltons) gene transcripts/gene/hr t r a n s c r i p t s per hr pDt5 5.9 X 10 6 777 . 10.3 140 6.4 X 1 0 - 1 1.00 X 10-10 pDtl7R 5.1 X 10 6 777 3.5 84 4.0 X 1 0 - 1 1.14 X i o - i ° pDtH16#5 2.4 X 10 6 777 2.0 52 3.8 X 1 0 _ 1 1.80 X 10-10 PDtA16-2#l 2.1 X 10 6 777 1.5 38 3.1 X 1 0 _ 1 1.90 X 10-10 pDtH16#l 2.4 X 10 6 774 0.6 30 1.5 X 1 0 _ 1 1.45 X 10-10 pDtA16-2#3 2.1 X 10 6 774 0.2 6 4.1 X l O " 2 1.40 X 10-10 pDt73 6.0 X 10 6 474 - - - -10-10 pDt73x27 2.3 X 10 6 474 0.1 9 4.7 X 10-2 1.90 X The values for the V m a x , and Km apparent were determined from double r e c i p r o c a l plots analogous to Lineweaver-Burk p l o t s . 136 plasmid pDt5) and 1.9 x 10 *° M (for plasmid pDtA16-2#l). The base pair v a r i a t i o n s found within the c o l l e c t i o n of genes in conjunction with the accompanying changes in the 5' flanking sequences such as found i n plasmids pDtH16#l (tRNA,"); pDtA16-2#3 (tRNA,") and 774 774 Ser pDt73x27 (tRNA^^) also resulted in minimal Kmapp changes when compared with each other or with pDt5. Unlike Kmapp, the i n i t i a l rates and maximum v e l o c i t i e s of the t r a n s c r i p t i o n reaction were strongly influenced by the differences between Sej? the tRNA genes. A comparison of the Vmax of plasmids pDt5, pDtl7R, pDtl6H#5 and pDtl6A-2#l suggested the e f f e c t of the flanking sequences. Ser These four plasmids contain i d e n t i c a l tRNA^^ genes but nonhomologous flanking sequences. The values f or Vmax varied by as much as 2-fold among the group of genes. The influence of the DNA 5' to the tRNA gene was demonstrated by the drop in the Vmax of the tRNA^^ precursor synthesis when 307 base pairs of 5' flanking sequences i n plasmid pDtl6H#l were deleted. This de l e t i o n (plasmid pDtl6A-2#3) retained only 36 bp of Drosophila DNA 5' to the gene and the r e s u l t i n g Vmax was decreased 4.0 f o l d . The differences between the Vmax for the See tRNA^^^ genes and those f o r the allogenes may be due to changes within the coding region or in the flanking DNA sequences. On the other hand, since the a l t e r a t i o n s in the coding regions d i d not s i g n i f i c a n t l y a f f e c t the Kmapp, i t seems u n l i k e l y that these a l t e r a t i o n s would influence the template a c t i v i t y (Vmax). The data in Table I I I suggest, therefore, that the 5' flanking sequences modulate Vmax, the template a c t i v i t y , 137 without s i g n i f i c a n t l y a f f e c t i n g the Kmapp, the template a f f i n i t y f o r the enzyme complex. E. Tr a n s c r i p t i o n k i n e t i c s of plasmids containing more than one gene. Figures 31 to 33 showed that plasmids containing more than one gene, pDtl6, pDt27 and pDtBH27#l exhibited s i m i l a r saturation k i n e t i c s as single gene t r a n s c r i p t i o n reactions when transcribed i n Schneider II c e l l e xtracts. The k i n e t i c data obtained from t h i s analysis are summarized in Table IV. Since pDtl6, pDt27 and pDtBH27#l contain multiple tRNA genes, the t r a n s c r i p t i o n products of each gene could not be independently measured, thus, neither the Kmapp nor the Vs values f or each gene were determined. The Vs values shown in Table IV correspond to the i n i t i a l Ser rate of t r a n s c r i p t i o n of a l l the tRNA genes found in the three plasmids. Table V compares the k i n e t i c data obtained from the t r a n s c r i p t i o n of Ser Ser tRNA genes derived from pDtl6. The Vs determined f or both tRNA genes on pDtl6 (160 transcripts/gene/hr) was almost twice the sum of the normalized v e l o c i t i e s determined f or the i n d i v i d u a l recloned genes (82 transcripts/gene/hr f o r pDtH16#l and #5 and 44 transcripts/gene/hr f o r pDtA16#l and #3). The mechanism by which the two tRNA genes on pDtl6 increase the o v e r a l l rate of t r a n s c r i p t i o n i s not c l e a r . Since tRNA genes are found clustered i n the Drosophila genome, the proximity of one gene may a f f e c t the rate of expression of the next. In addition to c l u s t e r i n g the o r i e n t a t i o n of the tRNA with respect to d i r e c t i o n of t r a n s c r i p t i o n may 138 Figure 31. Tra n s c r i p t i o n of pDtl6 in the presence and absence of pBR322 DNA. Tran s c r i p t i o n reactions using pDtl6 DNA were c a r r i e d out in the absence (A) or the presence of pBR322 DNA added to keep the t o t a l DNA concentration at 1.0 yg per 50 y l reaction (•) and the products were analyzed as described in Methods. The data obtained from t r a n s c r i p t i o n in the presence and absence of pBR322 i s plo t t e d as the v e l o c i t y of the reaction (cpm/hr) versus the concentration of the template DNA (M). Lanes 1 to 6 in the inset show t r a n s c r i p t i o n products derived from pDtl6. 139 140 Figure 32. Transcription of pDt27 in the presence and absence of pBR322 DNA. Transcription reactions using pDt27 DNA were c a r r i e d out in the absence (A) or the presence of pBR322 DNA added to keep the t o t a l DNA concentration at 1.0 yg per 50 y l reaction (•) and the products were analyzed as described in Methods. The data obtained from t r a n s c r i p t i o n in the presence and absence of pBR322 i s plotted as the v e l o c i t y of the reaction (cpm/hr) versus the concentration of the template DNA (M). Lanes 1 to 6 in the inset show t r a n s c r i p t i o n products derived from pDt27. 141 142 Figure 33. Tra n s c r i p t i o n of pDtBH27#l in the presence and absence of PBR322 DNA. Tran s c r i p t i o n reactions using pDtBH27#l DNA were c a r r i e d out in the absence (A) or the presence of pBR322 DNA added to keep the t o t a l DNA concentration at 1.0 ug per 50 y l reaction (•) and the products were analyzed as described in Methods. The data obtained from t r a n s c r i p t i o n in the presence and absence of pBR322 i s plo t t e d as the v e l o c i t y of the reaction (cpm/hr) versus the concentration of the template DNA (M). Lanes 1 to 6 in the inset show t r a n s c r i p t i o n products derived from pDtBH27#l. 143 Table IV Kinetic parameters from i n v i t r o t r a n s c r i p t i o n of plasmids . . Ser containing more than one tRNA gene Plasmid MW of Type of Vs Vs DNA tRNA S e r -pBR +pBR (daltons) gene transcripts/gene/hr pDtl6 7.4 x 10° 777/774 575 160 pDt27 6.9 x 10 6 444/444* 1.9 103 pDtBH27#l 2.6 X 10 6 444/444 1.3 51 also contains 4 tRNA genes 145 Table V Comparison of the k i n e t i c parameters from the jj i v i t r o Sex? t r a n s c r i p t i o n of the tRNA genes derived from pDtl6 Plasmid Type of tRNA S e r gene Vs Vs -pBR +pBR transcripts/gene/hr Kmapp M PDtl6 pDtH16#l pDtH16#5 pDtA16#l pDtA16#3 777/774 774 777 777 774 5.0 0.6 2.0 1.5 0.2 160 30 52 38 6 1.45 x 10 -10 1.80 x 10 -10 1.40 x 10 -10 1.90 x 10 -10 146 also influence expression. Further experimentation i s necessary to explore these p o s s i b i l i t i e s . . . Ser Val F. Tr a n s c r i p t i o n competition between tRNA genes and a tRNA gene I f the k i n e t i c s of t r a n s c r i p t i o n are Michaelis-Menten type then I would expect that the competition between two templates to follow standard competitive i n h i b i t i o n curves. To examine t h i s p r e d i c t i o n the competition Ser V a l between pDt5 (tRNA 7 ? ?) and pDt0.3 (tRNA* ; 144) was followed by measuring the amounts of t r a n s c r i p t formed by each of the two genes when both were present i n the reaction mixture. The product from Ser* t r a n s c r i p t i o n of pDt0.3 was smaller than that from the tRNA genes thus allowing simultaneous monitoring by polyacrylamide gel electrophoresis. In keeping with the experimental design f o r measuring competitive i n h i b i t i o n , the concentration of one gene (designated the competitor gene) was kept constant, while the concentration of the other gene (designated the substrate gene) was varied. Steady state k i n e t i c measurements (as described above) were performed i n the absence of competitor DNA to determine the k i n e t i c parameters Vmax and Kmapp for each template (Table VI). Addition of pDt5 as the competitor DNA template at a f i n a l concentration of 0.1 or 0.2 ug per t r a n s c r i p t i o n reaction along with increasing concentrations of the substrate DNA (pDt0.3) a l t e r e d the Kmapp as expected f o r competitive i n h i b i t i o n (Figure 35). The Kmapp for the Val tRNA^ gene increased in the presence of the competitor from 1.5 x 1 0 - 1 0 M to 3.8 x 1 0 - 1 0 M for 0.1 ug of competitor and to 147 Figure 34. T r a n s c r i p t i o n competition between a tRNA^^ gene and a Val tRNA gene. Competition experiments were performed using a t o t a l DNA concentration of 1.0 yg in the reaction. For each reaction, precursor tRNAs synthesized from both the competitor and substrate templates were monitored as described in the Methods. An autoradiograph representing the electrophoretic separation of l a b e l l e d RNAs derived from pDt0.3 and pDt5 is shown in Figure 34A. Lanes 1-5 show the t r a n s c r i p t i o n product of 0.01 - 0.07 yg of pDt5. Lanes 6-15 show the t r a n s c r i p t i o n products from both pDt5 as substrate DNA and 0.1 yg (lanes 6-10) and 0.2 yg (lanes 11-15) of pDt0.3 DNA as competitor DNA. The Cerenkov r a d i a t i o n in the t r a n s c r i p t i o n products was determined as described in the Methods and was plotted as the r e c i p r o c a l of the reaction v e l o c i t y (cpm/hr) 1 for the substrate gene against the r e c i p r o c a l of the substrate concentration i n the reaction (M . Since competition reactions contain a constant amount of competitor DNA and increasing concentrations of substrate DNA, a value of Ki can be e m p i r i c a l l y determined by the s h i f t i n the Kmapp f o r the substrate DNA. Figure 34B: pDt5 as the substrate DNA and 0.1 yg (o); 0.2 yg (•) or no (•) pDt0.3 DNA as competitor. pre tRNAf^ pre tRNA V^' 4> CO 149 150 Figure 35. Tr a n s c r i p t i o n competition between a tRNA gene and a Ser tRNA^y^ gene. Competition experiments were performed using a t o t a l DNA concentration of 1.0 yg in the reaction. For each reaction precursor tRNAs synthesized from both the competitor and substrate templates were monitored as described i n the Methods. An autoradiograph representing the electrophoretic separation of l a b e l l e d RNAs derived from pDt0.3 and pDt5 is shown in Figure 35A. Lanes 1-5 show the t r a n s c r i p t i o n product of 0.01 - 0.07 yg of pDt0.3. Lanes 6-15 show the t r a n s c r i p t i o n products from both pDt0.3 as substrate DNA and 0.1 yg (lanes 6-10) and 0.2 yg (lanes 11-15) of pDt5 DNA as competitor DNA. The Cerenkov r a d i a t i o n in the t r a n s c r i p t i o n products was determined as described i n the Methods and was plotted as the r e c i p r o c a l of the reaction v e l o c i t y (cpm/hr) for the substrate gene against the r e c i p r o c a l of the substrate concentration in the reaction (M "S . Since competition reactions contain a constant amount of competitor DNA and increasing concentrations of substrate DNA, a value of Ki can be e m p i r i c a l l y determined by the s h i f t in the Kmapp for the substrate DNA. Figure 35B: pDt0.3 as the substrate DNA and 0.1 yg (•); 0.2 yg (•) or no (A) pDt5 DNA as competitor. 152 153 Figure 36. T r a n s c r i p t i o n competition between a tRNA^^ gene and a Val tRNA gene. Competition experiments were performed using a t o t a l DNA concentration of 1.0 yg in the reaction. For each reaction, precursor tRNAs synthesized from both the competitor and substrate templates were monitored as described in the Methods. An autoradiograph representing the electrophoretic separation of l a b e l l e d RNAs derived from pDt0.3 and pDt73X27 i s shown in Figure 36A. Lanes 1-6 show the t r a n s c r i p t i o n product of 0.01 - 0.6 yg of pDt0.3. Lanes 7-24 show the t r a n s c r i p t i o n products from both pDt0.3 as substrate DNA and 0.1 yg (lanes 7-12), 0.2 yg (lanes 13-18) and 0.3 yg (lanes 19-24) of pDt73X27 DNA as competitor DNA. The Cerenkov r a d i a t i o n in the t r a n s c r i p t i o n products was determined as described in the Methods and was plotted as the r e c i p r o c a l of the reaction v e l o c i t y (cpm/hr) - 1 for the Bubstrate gene against the r e c i p r o c a l of the substrate concentration in the reaction (M "S . Since competition reactions contain a constant amount of competitor DNA and increasing concentrations of substrate DNA, a value of Ki can be e m p i r i c a l l y determined by the s h i f t in the Kmapp for the substrate DNA. Figure 36B: pDt0.3 as the substrate DNA and 0.1 yg (o); 0.2 yg (•); 0.3 yg (r) or no (A) pDt73x27 DNA as competitor. 155 156 Figure 37. Transcription competition between a tRNA^^ gene Val without a terminator sequence and a tRNA gene. Competition experiments were performed using a t o t a l DNA concentration of 1.0 yg in the reaction. For each reaction, precursor tRNAs synthesized from both the competitor and substrate templates were monitored as described in the Methods. An autoradiograph representing the electrophoretic separation of l a b e l l e d RNAs derived from pDt0.3 and pDt73 is shown in Figure 37A. Lanes 1-18 show the t r a n s c r i p t i o n products from pDt0.3 as substrate DNA and 0.1 yg (lanes 1-6), 0.2 yg (lanes 7-12) and 0.3 yg (lanes 13-18) of pDt73 DNA as competitor DNA. Lanes 19-24 show the t r a n s c r i p t i o n product of 0.01 - 0.6 yg of pDt0.3. The Cerenkov r a d i a t i o n in the t r a n s c r i p t i o n products was determined as described in the Methods and was p l o t t e d as the r e c i p r o c a l of the reaction v e l o c i t y (cpm/hr) 1 for the substrate gene against the r e c i p r o c a l of the substrate concentration in the reaction (M "*"). Since competition reactions contain a constant amount of competitor DNA and increasing concentrations of substrate DNA, a value of Ki can be e m p i r i c a l l y determined by the s h i f t i n the Kmapp for the substrate DNA. Figure 37B: pDt0.3 as substrate DNA and 0.1 yg (0); 0.2 yg (A); 0.3 yg (•) or no (o) pDt73 DNA as competitor. 8 CO CM o CM * $2 T— CM 0) oo m CO CM 158 159 Figure 38. E f f e c t of pDt5 DNA on the t r a n s c r i p t i o n of pDt0.3 DNA. Synthesis of the product from the competitor DNA template, pDt0.3, was monitored in competition experiments described in Figure 34. The r e c i p r i c a l of the t r a n s c r i p t i o n rate of pDt0.3 DNA (cpm/hr) ^ i s pl o t t e d as a function of the pDt5 template concentration (competitor gene) for two concentrations of pDt0.3. (•: 0.1 yg; A: 0.2 yg of DNA). 160 161 Figure 39. E f f e c t of pDt0.3 DNA on the t r a n s c r i p t i o n of pDt5 DNA. Synthesis of the product from the competitor DNA template, pDt5, was monitored in competition experiments described i n Figure 35. The r e c i p r o c a l of the t r a n s c r i p t i o n rate of pDt5 DNA (cpm/hr) 1 i s plotted as a function of the pDt0.3 template concentration (competitor gene) f o r two concentrations of pDt5. (•: 0.1 yg; A: 0.2 yg of DNA). 162 0 1 2 3 4 [Compet i to r gene] , nM 163 Figure 40. E f f e c t of pDt0.3 DNA on the t r a n s c r i p t i o n of pDt73X27 DNA. Synthesis of the product from the competitor DNA template, pDt73X27, was monitored in competition experiments described in Figure 36. The r e c i p r o c a l of the t r a n s c r i p t i o n rate of pDt73X27 (cpm/hr) * i s plotted as a function of the pDt0.3 template concentration (competitor gene) for three concentrations of pDt73X27. (o: 0.1 yg; A: 0.2 yg; t : 0.3 yg of DNA). 164 1 1 1 1.0 2.0 3.0 4.0 [Competitor gene] , nM 165 Table VI Comparison of K i , Kmapp and Vmax Plasmid Type of Vmax Km apparent Ki tRNA gene pmoles t r a n s c r i p t / h r pDt5 o 777 Ser 6. .4 x l O " 1 1.0 X l O " 1 0 * 1, ,9 X l O " 1 0 pDt73 + „ 474 Ser - - 1, .6 X l O " 9 pDt73x27 + c 474 Ser 4. .7 x l O " 2 1.9 X 10- 1 0M 5. .2 X l O " 1 0 pDtO.3 V a l 4 8. .0 x l O " 1 1.5 X 10- 1 0M 1, ,8 X l O " 1 0 + K i ' s f o r pDt73 and pDt73x27 were determined from competition experiments as described in Figures 36, 37 and 40 except that the concentration of competitor templates were 0.1, 0.2 and 0.3 yg. 166 7.0 x 10 M in the presence of 0.2 yg of the competitor template (Table VI). The Vmax of the reaction was unaffected by the addition of the competitor DNA as would be expected f o r pure competitive i n h i b i t i o n (Figure 35A). In the r e c i p r o c a l experiment, a greater s h i f t i n the Kmapp was observed when pDt0.3 was used as a competitor (Figure 34). The Kmapp of the plasmid pDt5 increased from 1.0 to 10 1 0 M to 4.5 x 1 0 - 1 ° M in the presence of 0.1 yg of competitor DNA and to 8.0 x 1 0 - 1 0 M in the presence of 0.2 yg of pDt0.3 (Table VI). Analyzed by Michaelis-Menten k i n e t i c s , the s h i f t i n the Kmapp caused by the addition of a second template to the t r a n s c r i p t i o n mixture can be used to measure K i , the i n h i b i t i o n constant. I define Ki as the competitive a b i l i t y of the competitor DNA and the value of Ki can be obtained from the mathematical expression r e l a t i n g the Kmapp, the concentration of i n h i b i t o r DNA, the Kmapp s h i f t and Ki (142). Evaluated from the data i n Figure 34 and Figure 35, the Ki was found to be 1.8 x 1 0 _ 1 ° for pDt0.3 and 1.9 x 1 0 - 1 ° for pDt5. The Ki was also determined in another way. The e f f e c t of increasing concentrations of the substrate DNA on the t r a n s c r i p t i o n of competitor DNA was followed by determining the amount of competitor product synthesized. The r e c i p r o c a l of the rate of t r a n s c r i p t i o n of the competitor template was plot t e d versus the substrate DNA concentration f o r each of the two competitor concentrations. The r e s u l t s (shown in Figures 38) were two int e r s e c t i n g s t r a i g h t l i n e s . The point of i n t e r s e c t i o n , which i s a -10 -10 measure of Ki (142), was at 1.7 x 10 and 1.8 x 10 f o r pDt0.3 and pDt5 re s p e c t i v e l y . The values determined by the r e c i p r o c a l plots are 167 almost i d e n t i c a l to those determined in the plots on Figures 38 and 39. I note that the ordinate to the point of i n t e r s e c t i o n of the l i n e s in Figures 38-40, which i s a measure of the Vmax for pDt5 and pDt0.3, corresponded exactly to the values determined from saturation curves. The k i n e t i c constants from i n h i b i t i o n experiment in which pDt73 and pDt73x27 were used as competitor DNAs and pDt0.3 was used as the substrate template are shown in Figures 36 and 37. The re s u l t s are summarized in Table VI. The plasmid pDt73 lacked the oligo-thymidylate sequence necessary f o r termination of t r a n s c r i p t i o n by RNA polymerase I II (73-75). In my t r a n s c r i p t i o n assay, pDt73 d i d not act as a template f o r RNA synthesis. Since there was no detectable RNA product d i r e c t e d by pDt73, I could not make k i n e t i c measurements on the plasmid. Plasmid pDt73x27 was Ser constructed to contain a 5' sequence i d e n t i c a l to the tRNA^^, gene Ser from pDt73 and a 3* flanking sequence from a tRNA^,^ gene from pDt27, including the termination s i g n a l . The reconstructed plasmid pDt73x27 dire c t e d a low l e v e l of tRNA precursor synthesis. Since pDt73x27 supported t r a n s c r i p t i o n , I was able to perform k i n e t i c measurements on the plasmid (Figure 36, Table VI). Using pDt73 and pDt73x27, I investigated the e f f e c t of the termination sequence of the i n t e r a c t i o n of the t r a n s c r i p t i o n complex and competitor template by measuring the Ki (Figure 36 and 37). The re s u l t s of the competitive i n h i b i t i o n studies revealed the Ki for pDt73 was approximately 9-fold higher than that determined f o r pDt5 (Table VI). Addition of the terminator sequence from pDt27 to pDt73 increased the s t a b i l i t y of the t r a n s c r i p t i o n complex formed with the gene 3 f o l d (the Ki 168 decreased 3 f o l d ) . However, the Ki determined for the plasmid pDt73x27 was s t i l l 3-fold higher than was the Ki determined for pDt5. As an aside, the r e c i p r o c a l of the t r a n s c r i p t i o n rate of pDt73x27 p l o t t e d as a function of the concentration of pDt0.3 added to the reaction (Figure 40) yielded a Ki of 1.7 x 10 ^ for pDt0.3, in exact agreement with the values determined from competition experiments with pDt5. G. Kinetics of t r a n s c r i p t i o n : Time course assay Since the t r a n s c r i p t i o n rate of d i f f e r e n t plasmids containing tRNA genes varied dramatically with template DNA concentration, i t was necessary to investigate the time course of the t r a n s c r i p t i o n Val reaction. Rajput demonstrated that t r a n s c r i p t i o n of tRNA genes in Drosophila Schneider II c e l l free extracts exhibited biphasic k i n e t i c s of Ser* product synthesis (144). S i m i l a r l y , the k i n e t i c s of tRNA gene t r a n s c r i p t i o n in Schneider II c e l l free extracts containing pBR322 DNA to remove the e f f e c t of the t r a n s c r i p t i o n i n h i b i t o r were biphasic. The time course of pDt5 t r a n s c r i p t i o n i s shown in Figure 41. A d i s t i n c t lag period during which a low rate of RNA synthesis was observed l a s t i n g approximately 20 min. Following the lag'period, RNA synthesis was much Ser more rapid. Active tRNA gene t r a n s c r i p t i o n was l i n e a r and continued for at l e a s t an a d d i t i o n a l 80 min. These type of k i n e t i c s has been observed in a number of t r a n s c r i p t i o n systems (47, 54, 62, 110, 111). The lag i n a product formation has been interpreted to be the time required for the formation of a multicomponent t r a n s c r i p t i o n complex. 169 Figure A l . T r a n s c r i p t i o n time course assay. In Figure 41A t r a n s c r i p t i o n reactions containing 0.1 ug pDt5 DNA and 0.9 yg pBR322 DNA were performed and analyzed as described in the Methods, except that reactions were terminated at indicated times. Reactions in lanes 1-10 were stopped a f t e r 5, 10, 15, 20, 25, 30, 45, 60, Ser 90 and 110 min. of incubation, r e s p e c t i v e l y . Total tRNA t r a n s c r i p t i o n for each time point was determined by summing the Cerenkov Ser Ser ra d i a t i o n i n both precursor tRNA (pre-tRNA^^^) and mature size Ser RNA (tRNA 7 7 7> in each band. Figure 41B shows an autoradiogram of the denaturing polyacrylamide gel in which t r a n s c r i p t s were separated. 170 T 1 1 1 1 1 1 1 1 1 r 2 0 4 0 6 0 80 100 Time ( m i n ) of T r a n s c r i p t i o n 171 172 H. Kinetics of t r a n s c r i p t i o n : Enzyme concentration curve To determine whether any of the factors i n the extract involved in the formation of a multicomponent complex recycled during the many rounds of t r a n s c r i p t i o n , I examined the e f f e c t of extract concentration on the Ser* o v e r a l l t r a n s c r i p t i o n process. The rate of tRNA^ 7 gene (pDt5) t r a n s c r i p t i o n p l o t t e d as a function of amount of extract added to the reaction i s shown in Figure 42. At low extract concentration (5 y l extract/50 y l reaction) there was a low rate of tRNA synthesis. As the amount of extract added to the reaction increased, an increasing proportion of t r a n s c r i p t i o n a c t i v i t y was observed as r e f l e c t e d by an upward curvature of the extract concentration curve. At higher concentrations of extract (15-25 y l extract/50 reactions) the rate of t r a n s c r i p t i o n became proportional to the amount of extracted added. Although the amount of pDt5 DNA (0.2 yg) in each reaction was constant, pBR322 DNA was added to t r a n s c r i p t i o n reactions containing increasing amounts of extract to remove the e f f e c t of the t r a n s c r i p t i o n i n h i b i t o r . The shape of the extract concentration curve i s t y p i c a l f o r a r e c y c l i n g component. I. K i n e t i c s of t r a n s c r i p t i o n : Extract dependence As shown in Figure 43 the i n i t i a l and f i n a l v e l o c i t y of t r a n s c r i p t i o n of pDt0.3 was dependent on the extract preparation used to support t r a n s c r i p t i o n whereas the Kmapp for pDt0.3 was not. Comparing the le a s t active extract preparation 922 with the most active 118, the Vs f o r pDt0.3 increased from 65 transcripts/gene/hr to 130 transcripts/gene/hr and the 173 Figure 42. Extract concentration curve. T r a n s c r i p t i o n reactions containing 0.2 yg pDt5 were performed and analyzed as described i n the Methods except that the amount of extract added to the reaction was varied. Reactions i n lanes 1 to 5 in the inset were c a r r i e d out with 5 y l , 10 y l , 15 y l , 20 y l , and 25 y l of Drosophila Schneider II c e l l extract. The s a l t concentration i n the mixture was maintained by adding 0 to 20 y l of buffer containing 30 mM T r i s - C l pH 7.9, 120 mM KCI, 5 mM MgCl 2 and 0.5 mM MgCl 2 and 0.5 mM d i t h i o t h r e i t o l . To eliminate the e f f e c t of the t r a n s c r i p t i o n i n h i b i t o r the t o t a l DNA concentration i n the reaction mixture was maintained at 0.04 Ser yg/yl extract by adding pBR322 DNA. To t a l tRNA synthesis was analyzed as described in the Methods. 174 Volume ( JJI ) of Extract 175 Figure A3. Extract dependence on t r a n s c r i p t i o n . In Figure 43A t r a n s c r i p t i o n reactions containing varying concentrations of pDt0.3 DNA were performed and analyzed as described in the Methods. Three d i f f e r e n t extracts, 922 ( i ) , 930 ( I ) and 118 (•) were used to analyze the e f f e c t of extract on the k i n e t i c s of t r a n s c r i p t i o n . The values for the k i n e t i c parameters are summarized in Table VII. Figure 43B shows an autoradiogram in which t r a n s c r i p t s synthesized in 118 (lanes 1 to 6), 930 (lanes 7 to 12), 922 (lanes 13 to 18) were separated on a denaturing polyacrylamide g e l . 176 7 8 9 1 0 11 1 2 1 3 1 4 1 5 1 6 1 7 1 8 178 Table VII Kinetic parameters measured in d i f f e r e n t extracts Extract Preparation Kmapp (M) Vmax (cpm/hr) Vs (transcripts/gene/hr) 930 922 118 31700 18400 47600 98 65 130 179 Vmax increased from 18400 cpm/hr to 47600 cpm/hr while the Kmapp changed s l i g h t l y from 1.4 x 1 0 _ 1 ° M to 1.9 x 1 0 ~ 1 0 M (Table VII). T r a n s c r i p t i o n reactions performed with extract preparation 930 yielded near average values. A l l t r a n s c r i p t i o n reactions reported in t h i s thesis have been performed with extract preparation 930. 180 Discussion Ser I. Transcription of Drosophila tRNA genes High speed (100,000 x g) supernatant f r a c t i o n s derived from Drosophila Schneider II cultured c e l l s were employed to investigate the Sec* t r a n s c r i p t i o n a l and k i n e t i c properties of Drosophila tRNA genes. Similar extracts have been prepared by other investigators from other sources (47, 60-65). Early investigations by Rajput et a l . characterized the c a t a l y t i c properties of t h i s S-100 extract with respect to the divalent cation requirement, the i o n i c strength optima and the r e l a t i v e Val a c t i v i t i e s with d i f f e r e n t tRNA genes (137, 144). The a b i l i t y of crude extracts prepared from Drosophila Schneider II c e l l s to support Val accurate and s e l e c t i v e t r a n s c r i p t i o n of tRNA genes in p u r i f i e d templates were based on the following observations: a) plasmid vector d i d not d i r e c t s p e c i f i c t r a n s c r i p t i o n ; b) the major discr e t e t r a n s c r i p t s synthesized in response to tRNA genes hybridized to DNA containing the tRNA gene f i x e d to n i t r o c e l l u l o s e f i l t e r s ; c) the t r a n s c r i p t i o n products exhibited a precursor-product r e l a t i o n s h i p in which the l a r g e s t t r a n s c r i p t was processed to give to a series of smaller RNAs, the smallest of which corresponded in size to the mature form of the tRNA; d) the t r a n s c r i p t s recovered from polyacrylamide gels accounted for a large f r a c t i o n of the newly synthesized RNA. Moreover, v i r t u a l l y a l l of the RNA synthesized in v i t r o in response to the tRNA gene template was d i r e c t e d by RNA polymerase I I I . L i t t l e RNA polymerase I and RNA polymerase II a c t i v i t y were associated with t h i s extract. 181 Ser The jji v i t r o t r a n s c r i p t i o n of the cloned Drosophila tRNA genes (except pDt73) resulted in a d i s c r e t e series of RNAs reminiscent of the Val t r a n s c r i p t s derived from tRNA genes suggesting that the reactions containing e i t h e r set of tRNA gene exhibited s i m i l a r t r a n s c r i p t i o n and processing properties. The plasmid pDt73 yielded no s p e c i f i c RNA species when analyzed in these t r a n s c r i p t i o n reactions. Because the i n a b i l i t y of Ser the tRNA^y^ gene in pDt73 produce d i s c r e t e t r a n s c r i p t s was due to the lack of a termination s i t e 3' to the s t r u c t u r a l gene, i n s e r t i o n of a Ser termination s i t e from a tRNA^^ gene (pDt27) into the 3' portion of Sec the tRNA^^ gene in pDt73 resulted in s p e c i f i c RNA synthesis (pDt73x27). As shown in Figure 20 the s i z e analysis of the t r a n s c r i p t i o n Sec products from the other tRNA genes revealed that the primary RNA species was considerably larger than the mature form of the tRNA (perhaps representing the precursor tRNA) while the smallest RNA species corresponded in s i z e to the mature tRNA and was represented in a l l t r a n s c r i p t i o n s . Table II summarizes the s i z e of the t r a n s c r i p t i o n products. These r e s u l t s together with the h y b r i d i z a t i o n analysis in Val Figure 21 established that in addition to tRNA genes Drosophila Ser Schneider II c e l l - f r e e extracts s e l e c t i v e l y transcribe tRNA genes. Although the processing experiments were not performed the Val precursor-product r e l a t i o n s h i p exhibited by tRNA gene products argues Sec that t h i s r e l a t i o n s h i p also exists for tRNA gene products. Since the predominant t r a n s c r i p t i o n products dir e c t e d by the Sec d i f f e r e n t tRNA genes were 20 - 25 nucleotides longer than the mature form of the tRNA the approximate s i t e s specifying t r a n s c r i p t i o n i n i t i a t i o n 182 and termination could be determined. The DNA sequence analysis of a l l the tRNA templates except pDt73 showed the presence of a c l u s t e r of 4 - 9 T residues on the noncoding strand, 16 - 20 nucleotides from the 3' end of each gene. Because termination of t r a n s c r i p t i o n by RNA polymerase I I I i s dependent on a stretch of at l e a s t 4 T residues (73-75), the s i t e s of t r a n s c r i p t i o n i n i t i a t i o n are estimated to be about 4 - 7 bp upstream from the s t a r t of the coding element. The synthesis of some la r g e r minor t r a n s c r i p t i o n products suggest that other i n i t i a t i o n s s i t e s 6 - 8 bp further upstream are employed at reduced e f f i c i e n c y (see Figure 20). Heterogeneity in precursor size may be due heterogenous termination of t r a n s c r i p t i o n in a large c l u s t e r of T residues. I I . Drosophila Schneider II c e l l - f r e e extracts contain an i n h i b i t o r As in early studies, which analyzed the molecular mechanisms responsible for regulating eukaryotic Class III genes (61,63,79,97,112), I have also employed a k i n e t i c approach of the analysis. However, unlike the other investigators, I was able to make accurate quantitative k i n e t i c Ser measurements on a series of tRNA genes transcribed by RNA polymerase III in a homologous jln v i t r o c e l l - f r e e system. While e s t a b l i s h i n g a r e l i a b l e competition assay for adenovirus VA RNA genes in Hela c e l l - f r e e extracts, Fowlkes and Shenk (63) observed that the addition of pBR322 DNA to the t r a n s c r i p t i o n reactions stimulated synthesis Arg of both VAI and VAII RNA. S i m i l a r l y , t r a n s c r i p t i o n of a tRNA gene in Drosophila Kc c e l l - f r e e extracts was stimulated by the presence of pBR322 (110). These observations suggested the presence of an i n h i b i t o r in both 183 Hela and Drosophila Kc c e l l - f r e e extracts which could conceivably be bound by pBR322 DNA. In my analysis, the rate of t r a n s c r i p t i o n of tRNA genes as a function of template DNA input in Drosophila Schneider II extract, also indicated the presence of an i n h i b i t o r which was removed by higher DNA concentrations. The addition of nontemplate DNA such as pBR322 or the synthetic copolymer poly dG.C to the reaction mixtures l e d to the r e l i e f of the i n h i b i t o r e f f e c t . The amount of DNA required to r e l i e v e i n h i b i t i o n was the same as the concentration of template DNA required to obtain maximum a c t i v i t y in the absence of nontemplate DNA. The addition of non-template DNA to the t r a n s c r i p t i o n mixture revealed that the saturation of the enzyme complex in the reaction occurred at much lower l e v e l s than previously suspected and that the rate of t r a n s c r i p t i o n of template DNA was proportional to the number of genes in the reaction. However, not a l l of deoxyribonucleotides e f f e c t i v e l y removed the i n h i b i t o r a c t i v i t y on t r a n s c r i p t i o n . The synthetic copolymer poly dA.T, single stranded DNA and the single stranded DNA analogue heparin i n h i b i t e d t r a n s c r i p t i o n to varying degrees. While these DNAs or DNA analogues may bind the i n h i b i t o r as w e l l , they would appear to sequester e s s e n t i a l components of the t r a n s c r i p t i o n complex. Synthesis of the tRNA precursors was p a r t i c u l a r l y s e n s i t i v e to heparin. Transcription complexes which were preincubated up to 40 min. with template DNA were s t i l l s e n s i t i v e to heparin i n h i b i t o r . Apparently, heparin can either invade the t r a n s c r i p t i o n complex and i n a c t i v a t e i t or bind some factors which recycle during the consecutive rounds of t r a n s c r i p t i o n s (50, 106). The e f f e c t of poly dA.T suggests that the components of the t r a n s c r i p t i o n complex 184 p r e f e r e n t i a l l y bind either to AT r i c h DNA sequences or to DNAs which are singl e stranded (poly dAT can e a s i l y be denatured due to i t ' s low melting temperature). The nature of the t r a n s c r i p t i o n i n h i b i t o r and i t ' s a c t i v i t y in the reaction are not cl e a r . I t i s doubtful that the i n h i b i t o r plays an important r o l e i n Class III gene regulation since i t i s e a s i l y removed by nonspecific DNA. Evidence presented here suggest that the i n h i b i t o r i s a DNA binding protein since i t was s e l e c t i v e l y removed by the addition of pBR322 and poly dG.C DNA. The presence of an i n h i b i t o r in three S-100 extracts i s o l a t e d from d i f f e r e n t sources indicates that i t may be a general phenomenon (63,110). I l l . K i netics of t r a n s c r i p t i o n i n the presence of non template DNA Ser* Because the t r a n s c r i p t i o n of tRNA genes in the presence of nontemplate pBR322 DNA exhibited standard Michaelis-Menten dependence on the concentration of DNA template with a high degree of accuracy, I followed the i n t r i c a t e processes of the t r a n s c r i p t i o n reaction by k i n e t i c analysis. By employing the rate equation f o r a one-substrate enzyme catalyzed reaction, I determined the k i n e t i c parameters of the t r a n s c r i p t i o n of each Sezr tRNA gene; the apparent a f f i n i t y of the t r a n s c r i p t i o n complex for the template DNA, the Kmapp, the i n i t i a l v e l o c i t y of the reaction, Vs, and the maximum v e l o c i t y of the reaction, which was interpreted as template a c t i v i t y , Vmax. The importance of performing k i n e t i c analyses are two f o l d : 1) since factors which influence the rate of t r a n s c r i p t i o n may exert t h e i r e f f e c t s by either a f f e c t i n g the formation of the t r a n s c r i p t i o n 185 complex or the rate of stable t r a n s c r i p t i o n complex breakdown, determination of an apparent Km and Vmax allowed me to independently investigate these two parameters; and 2) the set of cloned Drosophila Sec melanogaster tRNA genes (some members share i d e n t i c a l intragenic sequences, but d i f f e r in the 5' and 3' flanking sequences while others d i f f e r in both point mutations within the s t r u c t u r a l gene and in the flanking sequences) has provided me with the necessary templates to q u a n t i t a t i v e l y study the e f f e c t s of intragenic and flanking sequences in tRNA gene expression. The determination of the k i n e t i c parameters showed the Kmapp for a l l Ser —10 the tRNA genes varied only s l i g h t l y , ranging from 1.0 x 10 to 1.9 x 10 ^ M. In contrast, the Vmax changed s u b s t a n t i a l l y from 6.4 x -1 -2 10 pmoles of t r a n s c r i p t s / h r to 4.3 x 10 pmoles of t r a n s c r i p t s / h r . Sec A comparison of the i n i t i a l rates of t r a n s c r i p t i o n of tRNA^^ genes suggested that although intragenic sequences are e s s e n t i a l for complex formation, sequences flanking these i d e n t i c a l genes determine the rate of turnover of stable t r a n s c r i p t i o n complexes. The plasmids pDtH16#l and Set? Sec pDt73 containing the variant genes tRNA,,, and tRNA,,, 774 474 r e s p e c t i v e l y , also support very d i f f e r e n t rates of t r a n s c r i p t i o n in Drosophila Schneider II extracts with l i t t l e e f f e c t on the Kmapp when Sec compared to plasmids containing tRNA^^ genes. These two genes are Sec very s i m i l a r in structure to the tRNA^^ gene. They d i f f e r from the wildtype gene by t r a n s i t i o n mutations occurring at p o s i t i o n 16 (C/T) and p o s i t i o n 77 (A/G). The sequences flanking these genes are completely heterologous. The mutation at p o s i t i o n 77 which occurs in the amino 186 acceptor stem of the mature tRNA l i e s outside of the s p l i t control region e s s e n t i a l for the i n i t i a t i o n of t r a n s c r i p t i o n . I t i s u n l i k e l y that t h i s mutation a f f e c t s t r a n s c r i p t i o n e f f i c i e n c y or product s t a b i l i t y . A l l i s o n et a l . demonstrated that several point mutations located in the . Tvr aminoacceptor stem of a yeast SUP 4 tRNA J gene did not a f f e c t either the rate of mutant gene t r a n s c r i p t i o n or the s t a b i l i t y of the tRNA precursor (114). In addition, Rajput et a l . , showed that a mutation in Val the aminoacceptor stem [at p o s i t i o n 69 (A/G)] of a hybrid tRNA gene produced l i t t l e e f f e c t on t r a n s c r i p t i o n e f f i c i e n c y (137). These Ser observations suggest that the reduced i n i t i a l rate of tRNA^^ Ser synthesis when compared to the more active tRNA^^ genes i s l a r g e l y due to an i n h i b i t o r y e f f e c t exerted by the flanking sequences. Deletion Ser analysis of a tRNA 7 7^ gene provided evidence that i t i s the 5* flanking sequence which primarily determines the rate of tRNA precursor production from a t r a n s c r i p t i o n complex. Removal of the 5' flanking sequence to within 36 bp of the s t r u c t u r a l gene resulted in a 4.5 f o l d reduction in template a c t i v i t y without a f f e c t i n g the Kmapp. Similar r e s u l t s suggesting the 5'-flanking sequence dependence for e f f i c i e n t t r a n s c r i p t i o n have been reported for other tRNA genes (70, 116, 137); however, Schaak et a l . recently reported that a Kmapp for a Drosophila tRNA A r B gene was also affected by a d e l e t i o n of 5' flanking sequences (116). The apparent discrepancy between these data and mine i s for the moment unresolved. Ser The plasmid pDt73 which contains a tRNA^^ gene lacking a t r a n s c r i p t i o n termination sequence did not stimulate production of any 187 measurable RNA species in the t r a n s c r i p t i o n reaction. The i n a b i l i t y of t h i s gene to support detectable l e v e l s of tRNA synthesis seems to depend on sequences other than 3' flanking sequences, since some tRNA genes d e f i c i e n t in an o l i g o T t r a c t s t i l l d i r e c t near wild type rates of t r a n s c r i p t i o n (69, 99, 100, 110). When a termination sequence derived from pDt27 was l i g a t e d to pDt73, the r e s u l t i n g plasmid supported accurate but low l e v e l t r a n s c r i p t i o n . This low l e v e l of tRNA precursor synthesis could be a t t r i b u t e d to the C-T t r a n s i t i o n mutation within the D-control Ser region of the tRNA^ ? 4 gene's s p l i t promoter or to the 5' flanking sequence. Analysis of the consensus sequence of a tRNA gene promoter (69, 99, 100, 103, 109) indicated that the i n t e r n a l control region of the Sec Sec tRNA^^, gene and the tRNA ? 7 7 gene were v i r t u a l l y i d e n t i c a l . Furthermore, the differences between these two genes did not s i g n i f i c a n t l y a f f e c t the Kmapp. This suggests that these differences would be t o l e r a t e d with minimal e f f e c t on t r a n s c r i p t i o n e f f i c i e n c y . Examination of the 5' flanking sequence of pDt73 revealed an 11 bp a l t e r n a t i n g purine-pyrimidine sequence four nucleotides from the 5' end of the s t r u c t u r a l gene. A s i m i l a r sequence has been shown to dramatically reduce t r a n s c r i p t i o n of a Met va r i a n t tRNA gene in Xenopus germinal v e s i c a l extracts when positioned nine nucleotides upstream to the gene. Nordheim and Rich have shown that G-C r i c h a l t e r n a t i n g purine-pyrimidine t r a c t s 8-10 base pairs in length form Z-DNA in both supercoiled pBR322 and SV40 DNA (108). The presence of a r e l a t e d sequence at or near the t r a n s c r i p t i o n i n i t i a t i o n s i t e may a l t e r the DNA conformation such that the formation of an Ser i n i t i a t i o n complex i s hindered. I f the sequence in the tRNA^^^ 188 gene was e f f e c t i v e i n reducing the t r a n s c r i p t i o n rate, i t d i d not do so by a f f e c t i n g the a f f i n i t y of the t r a n s c r i p t i o n complex (the Kmapp). Ser* Taken as a whole, my data imply that the Kmapp of the tRNA genes i s dependent on the i n t e r n a l gene structure and not on the sequences flanking the gene. The s i m i l a r i t i e s between the Kmapp of the Ser V a l tRNA 7 7 7 and the tRNA^ genes probably r e s u l t from the high degree of sequence homology in the i n t e r n a l control regions. For example, Ser V a l the tRNA ? 7 7 gene (pDt 5) and the tRNA^ gene (pDt0.3) are homologous f o r 9 of the 12 nucleotides in the D-control region and f o r 9 of the 11 nucleotides i n the T-control region. A l l of the differences between these two genes f a l l i n places which are not s p e c i f i e d i n the consensus sequence (69, 99, 100, 103, 109). Unlike Kmapp, the v e l o c i t y of t r a n s c r i p t i o n as r e f l e c t e d in Vmax was dramatically affected by the sequences external to the gene. While the e f f e c t of the 5' flanking sequences has been documented before (50, 61, Ser 107, 116, 137), the separation of the k i n e t i c parameters f o r tRNA genes shows that 5' flanking sequences exert t h e i r influence on the breakdown of the t r a n s c r i p t i o n complexes, and not the s t a b i l i t y of the f i n a l complex. The influence of the 5' flanking sequence suggests that the i n t e r a c t i o n of these sequences with the t r a n s c r i p t i o n complex i s required f o r each round of product synthesis. This i n t e r a c t i o n may be somewhat analogous to b a c t e r i a l promoter systems in which the 5' flanking sequences would a f f e c t the formation of "open promoter complex" (1) and hence be involved in RNA chain i n i t i a t i o n . While I can only speculate on the nature of such i n t e r a c t i o n s , i t i s cl e a r that structures which might 189 a l t e r the DNA h e l i x , such as Z-DNA, could have strong adverse e f f e c t s on t r a n s c r i p t i o n . Moreover, 5' sequences r i c h in A-T or G-C residues could e f f e c t t r a n s c r i p t i o n by influencing the a b i l i t y of RNA polymerase to denature the sequences and therefore form an i n i t i a t i o n complex (an open promoter complex). F i n a l l y , i t i s i n t r i g u i n g to note that the 5' flanking region of pDt5 and pDt0.3 are t o t a l l y heterologous and yet both plasmids are transcribed at a very high rate. I t i s possible that high turnover rates can be stimulated by a v a r i e t y of d i f f e r e n t sequences (as can low turnover r a t e s ) . To date, there i s no d i r e c t evidence suggesting that p o s i t i v e or negative regulatory factors i n t e r a c t with 5' flanking sequences of tRNA genes thus influencing expression. The rates of RNA synthesis in these t r a n s c r i p t i o n reactions approach 3 nucleotides per second per enzyme complex. I f the radioactive UTP i s being d i l u t e d in s p e c i f i c a c t i v i t y i n the t r a n s c r i p t i o n reaction by endogenous l e v e l s of UTP, t h i s rate i s a lower l i m i t . While ijn vivo rates of tRNA synthesis have not been reported, the iii vivo rate of ribosomal RNA synthesis have been estimated at 10 nucleotides per sec (140). The in v i t r o system as analyzed here i s within a f a c t o r of 3 of t h i s i i i vivo rate. In assessing the s i g n i f i c a n c e of the Kmapp, i t i s important to Ser r e a l i z e that the time course of t r a n s c r i p t i o n of tRNA genes in Schneider II c e l l l i n e extracts e x h i b i t biphasic k i n e t i c s in which a 20 -30 min. lag in tRNA synthesis i s followed by a l i n e a r r a pid rate of t r a n s c r i p t i o n l a s t i n g f or at l e a s t 80 min (Figure 41). Similar k i n e t i c s have been observed in a v a r i e t y of t r a n s c r i p t i o n systems involving Class I I I genes (47, 60, 62, 110, 111). The lag in tRNA production has been 190 interpreted to represent the time required to assemble a multicomponent t r a n s c r i p t i o n complex (111, 137). During t h i s lag period, tRNA genes appear to r a p i d l y sequester a t r a n s c r i p t i o n f a c t o r ( s ) , present in the c e l l extract in l i m i t i n g concentrations (111). The binding of t h i s f a c t o r ( s ) which appears to be rapid r e s u l t s in a meta-stable complex not s u f f i c i e n t to promote rapid tRNA synthesis (111). The a d d i t i o n a l 10 - 30 min necessary to d i r e c t active t r a n s c r i p t i o n may involve complete t r a n s c r i p t i o n complex assembly or the conversion of the complex into an active form (see also 79). Recent studies involving the f r a c t i o n a t i o n of human and Xenopus c e l l extracts have implicated two t r a n s c r i p t i o n f a c t o r s , Factor IIIB and Factor IIIC, in the i n i t i a l stages of complex formation (50). Since the Kmapp for each gene were measured af t e r 90 min. of reaction, these values primarily r e f l e c t the a f f i n i t y of the c a t a l y t i c form of the t r a n s c r i p t i o n complex which ex i s t s during the l i n e a r portion of the reaction time course for DNA. Reconstitution of a p a r t i a l l y p u r i f i e d t r a n s c r i p t i o n system has provided evidence to suggest that a f a c t o r involved in the formation of the t r a n s c r i p t i o n complex (Factor IIIB) recycles during t r a n s c r i p t i o n . The dependence of the t r a n s c r i p t i o n rate on Drosophila Schneider II c e l l extract concentration as shown in Figure 42 implies that t r a n s c r i p t i o n factors in the reaction mixture recy c l e . I f the c e l l extract preparation contains a c a t a l y t i c subunit(s) then the amount of gene-stable t r a n s c r i p t i o n complexes which are in the t r a n s c r i p t i o n a l l y active form would increase at a nonlinear rate as the concentration of the extract increases. At low extract concentration the reaction rate was low and nonlinear. At high concentrations (of f a c t o r ( s ) 191 which recycles) the v e l o c i t y of the t r a n s c r i p t i o n reaction became proportional to the extract concentration suggesting that the factors in the reaction were in excess and therefore, not rate l i m i t i n g . I t i s possible that the Kmapp r e f l e c t s that part of the t r a n s c r i p t i o n complex which recycles. IV. Kinetic analysis of competing templates Ser Since the t r a n s c r i p t i o n of tRNA genes followed standard Hichaelis-Henten Kinetics I used competitive i n h i b i t i o n experiments to analyze the Ki of four genes. C l a s s i c a l competitive i n h i b i t i o n behavior was observed for each template i n the reaction. However, the Ki empir i c a l l y determined for a l l templates was found to be higher than the Kmapp for that template. The Ki i s defined as the d i s s o c i a t i o n constant f o r the formation of the enzyme-substrate complex. I f t r a n s c r i p t i o n reactions followed normal k i n e t i c s , the Ki determined should be equivalent to the d i s s o c i a t i o n constant of the RNA polymerase I I I (and factors) -gene complex and should be numerically le s s than or equal to the Kmapp. Since t h i s was not the case, the Ki's I have measured are not p r e c i s e l y the constant predicted by Michaelis-Menten k i n e t i c s . Like the values determined for the Kmapp, the s i g n i f i c a n c e of the Ki is revealed by the biphasic nature of the t r a n s c r i p t i o n k i n e t i c s . As described above, Kmapp pri m a r i l y r e f l e c t s the a f f i n i t y of the c a t a l y t i c form of the t r a n s c r i p t i o n complex f o r the DNA template. On the other hand in the competition experiments where the competing templates are added simultaneously to the reaction mixture, the Kis determined are probably 192 the d i s s o c i a t i o n constant for the rate l i m i t i n g step of complex assembly. The Ki r e f l e c t s the formation of the t r a n s c r i p t i o n complex during the lag phase of t r a n s c r i p t i o n and i s independent of the Kmapp values. The independence of Ki and Kmapp i s emphasized by the data i n Table VI where Ki i s seen to vary between template DNAs but Kmapp does not. Since the value of Ki i s larger than the corresponding Kmapp, the complex signaled by Ki i s less stable than the f i n a l form. Thus the f i r s t step i n t r a n s c r i p t i o n complex assembly which occurs during the lag phase of t r a n s c r i p t i o n although s u f f i c i e n t to y i e l d an exclusionary complex i n v i t r o i s further s t a b i l i z e d by eit h e r the addition of other components necessary to i n i t i a t e t r a n s c r i p t i o n or by assuming an active conformation. Comparison of the Ki's for pDt73 and pDt73x27 demonstrate the influence of the 3' flanking sequence on the formation of a stable complex. The addition of the terminator and surrounding sequences appeared to allow i n t e r a c t i o n with the t r a n s c r i p t i o n complex components which enhanced the s t a b i l i t y of the i n i t i a l stages 3-fold. The 3-fold difference in the Ki's for pDt5 and pDt73x27 suggests that sequences flanking the 5' end of the genes and perhaps i n t e r n a l point mutations Ser influence the formation of the exclusionary complex on tRNA genes. Similar conclusions have been reached about the flanking sequences from Arg Drosophila tRNA gene (116). The s i m i l a r Ki's observed for pDt0.3 and pDt5 suggests that the highly homologous intragenic control sequences have a s i m i l a r a f f i n i t y for an i n i t i a l component of the t r a n s c r i p t i o n complex. Several other groups have recently used competition between tRNA templates to compare the ef f e c t s of a l t e r a t i o n s of nucleotides in and 193 around the gene on the t r a n s c r i p t i o n properties. In short term t r a n s c r i p t i o n experiments such as those used by A l l i s o n et a l . (114), the maximum rate of t r a n s c r i p t i o n would be dominated by the Ki value, e s p e c i a l l y at DNA excess. Thus competitive strength would vary with o v e r a l l t r a n s c r i p t i o n rate, as was observed. The experiments reported by Sharp et a l . (110), are s i m i l a r to those presented in t h i s t h e s i s . Although the k i n e t i c parameters of the competing templates containing del e t i o n mutants were not determined by t h e i r experiments, the semi-quantitative data demonstrated the importance of the intragenic T-control region in complex s t a b i l i t y (110). The analysis reported here shows that Hichaelis-Menten k i n e t i c s can be used to examine the processes involved in tRNA synthesis i f nontemplate DNA are added to eliminate the e f f e c t of an endogenous i n h i b i t o r . By employing a k i n e t i c approach of t r a n s c r i p t i o n analysis on a series of Ser tRNA genes I was able to study independently the e f f e c t of intragenic and flanking sequences on eit h e r the formation or the turnover of preformed t r a n s c r i p t i o n complexes. The re s u l t s of t h i s analysis Ser demonstrate that v a r i a t i o n in the t r a n s c r i p t i o n rate of tRNA genes i s due to v a r i a t i o n in the rate of u t i l i z a t i o n of preformed complex and not due to a reduced template a f f i n i t y for the complex. Sequences upstream from the gene appear to int e r a c t with these complexes. I t i s t h i s i n t e r a c t i o n which would modulate the o v e r a l l rate of t r a n s c r i p t i o n . C l a s s i c a l competitive i n h i b i t i o n k i n e t i c s allowed the measurement of an i n h i b i t i o n constant. This analysis revealed a v a r i a t i o n in template a f f i n i t y f o r some i n i t i a l component of the t r a n s c r i p t i o n complex. The 194 formation of a stable t r a n s c r i p t i o n complex on a tRNA gene as measured by Ki i s not adequate to d i r e c t active product synthesis (since the t r a n s c r i p t i o n lag period l a s t s for 20-30 min.). V. Model for Transcription of tRNA genes The development of c e l l free extracts which support accurate t r a n s c r i p t i o n of Class I II genes on p u r i f i e d templates has provided valuable information about the nature and mechanism of action of the various components necessary f o r f a i t h f u l t r a n s c r i p t i o n . Analysis of t r a n s c r i p t i o n of mutant 5S RNA genes have demonstrated that a 34 nucleotide sequence within the s t r u c t u r a l gene i s s u f f i c i e n t to d i r e c t s p e c i f i c i n i t i a t i o n of t r a n s c r i p t i o n (77,78). In s i m i l a r studies tRNA genes and adenovirus VAI RNA genes are shown to contain two non contiguous i n t e r n a l control regions referred to as the D-control and the T-control regions (63,69,99,100,109). In addition, upstream sequences of Class I I I genes are found to modulate t r a n s c r i p t i o n e f f i c i e n c y (61,96,101,105,107, 116) while downstream sequences are involved in t r a n s c r i p t i o n termination (73-75). Together intragenic and flanking sequences of Class I II genes d i r e c t t r a n s c r i p t i o n complex formation, confer complex s t a b i l i t y and support t r a n s c r i p t i o n a c t i v i t y . Fractionation of the c e l l extracts has revealed that the c a t a l y t i c a c t i v i t y in extracts i s orchestrated by RNA polymerase III together with a number of protein t r a n s c r i p t i o n factors (50). Three f a c t o r s , Factor IIIA, Factor IIIB, and Factor IIIC, are involved in promoter recognition and 195 Figure 44. Model for t r a n s c r i p t i o n of tRNA genes. The f i v e stages in the model are described i n the text. 197 stable complex formation on Class I I I genes (50). Other factors not yet i d e n t i f i e d may be involved in regulating t r a n s c r i p t i o n . Factor IIIA i s a 5S RNA gene-specific factor of approximately 40,000 daltons (91). Complex formation on 5S RNA genes required both Factor IIIA and a factor common to a l l Class I I I genes, Factor IIIC (50). Factor IIIA binds to the intragenic control sequence of 5S RNA genes (113) as well as to 5S RNA (88). For t h i s reason, i t i s l i k e l y that the Factor IIIA gene-complex i s on the antisense strand. Recent f r a c t i o n a t i o n of yeast nuclei has revealed that Factor IIIC i s a large protein (M r 300,000) which stably binds to the T-control region of tRNA genes (124). Deletion studies in which 3' flanking sequences of a tRNA A r g gene were removed demonstrate that these sequences are important in Factor IHC-gene complex s t a b i l i t y (111,116). This i n f e r s an association between the 3* flanking sequences and Factor IIIC. Factor IIIB apparently functions to s t a b i l i z e Factor IHC-gene complexes on tRNA and VAI RNA genes but only a f t e r extended incubation (50). Factor IIIB does not remain stably bound. I t appears to recycle during complex formation. F i n a l l y , the formation of stable complexes on Class III genes in the absence of RNA polymerase I I I suggests that polymerase i s the f i n a l component in the assembly of the t r a n s c r i p t i o n complex. RNA polymerase recognition must involve both protein-protein and protein-DNA contacts in the complex. The current state of knowledge leads me to propose a model of tRNA gene t r a n s c r i p t i o n . The model shown in Figure 44 includes: a) t r a n s c r i p t i o n factors Factor IIIB (B) and Factor IIIC (C) and RNA polymerase I I I ; b) the D-control (D) and the T-control (T) intragenic 198 regions, the t r a n s c r i p t i o n i n i t i a t i o n s i t e ( i ) and termination s i t e ( t ) . Since the k i n e t i c s of t r a n s c r i p t i o n indicate a multistage process, the 5 stages in the model shown accommodate the following; a) t r a n s c r i p t i o n complex assembly, b) the s t a b i l i t y of the complexes, c) the f l e x i b i l i t y and s i t e s e l e c t i o n of t r a n s c r i p t i o n i n i t i a t i o n , d) the rate of complex u t i l i z a t i o n , e) t r a n s c r i p t i o n termination, and f) r e i n i t i a t i o n . Stage 1 Before the i n i t i a t i o n of t r a n s c r i p t i o n , p u r i f i e d templates r a p i d l y sequester a t r a n s c r i p t i o n factor in the c e l l extract to form a gene-stable complex (111). Formation of t h i s complex appears to require the i n t e r a c t i o n of Factor IIIC (C) p r i m a r i l y with the T-control region (T) of the tRNA gene (50, 124). Secondary interactions probably occur in the 3' regions of the gene as well as the 3' flanking sequences (111, 116). The large s i z e of Factor IIIC could e a s i l y accommodate these i n t e r a c t i o n s . The binding of Factor IIIC onto native templates may i n i t i a l l y involve double stranded DNA but i f the f i n a l complex i s to be t r a n s c r i p t i o n a l l y active and i f Factor IIIC remains stably bound for many rounds of t r a n s c r i p t i o n (50) then ultimately Factor IIIC must associate with single stranded DNA. The a f f i n i t y of Factor IIIC for single stranded DNA i s demonstrated by i t s a b i l i t y to bind to heparin Sepharose columns (124). By analogy to Factor IIIA t h i s i n t e r a c t i o n probably occurs on the antisense strand of the gene. The i n i t i a l complex formed with Factor IIIC on template DNA, i s not s u f f i c i e n t to promote rapid RNA synthesis but w i l l preclude the t r a n s c r i p t i o n of a competing template added subsequently to 199 the reaction i f a l l of Factor IIIC i s bound to the f i r s t template (111). Since the events leading to rapid RNA synthesis a f t e r Factor IIIC binding are slow and temperature dependent (111) they may involve completion of the multicomponent t r a n s c r i p t i o n complex or rearrangement of the Factor IHC-gene complex into an active conformation. Because reassembly of an active t r a n s c r i p t i o n complex at the beginning of each subsequent round of t r a n s c r i p t i o n complex does not require a lag period, I propose that i t i s the l a t t e r which i s rate determining in the i n i t i a l complex formation. This rearrangement into an active conformation may involve the t r a n s i t i o n between Factor IHC-double stranded and Factor IIIC-single stranded DNA gene complexes. The Ki measured in t h i s thesis could r e f l e c t Factor IIIC binding or Factor IIIC-Factor IIIB s t a b i l i z a t i o n (see below). Stage 2 L i t t l e i s known about Factor IIIB. Competition experiments have suggested the p o s s i b i l i t y of a D-control factor which binds to the D-control region independently of a T-control factor (Factor IIIC) (111). I have assumed that Factor IIIB i s the putative D-control f a c t o r and thus associates only with the D-control region. This association would then s t a b i l i z e the Factor IHC-gene complex perhaps by increasing the l e v e l of template DNA denaturation. In addition to confering complex s t a b i l i t y , Factor IIIB may play a r o l e in guiding the active s i t e of RNA polymerase III to the s i t e of i n i t i a t i o n on the template DNA. 200 Stage 3 RNA polymerase I I I , recognizing the Factor IIIB-Factor IHC-gene complex, must int e r a c t with t h i s complex and a t t a i n the proper active conformation. To achieve t h i s , RNA polymerase III might i n t e r a c t with the the sense strand of the gene while using the proteins Factor IIIB and Factor IIIC to p r e c i s e l y p o s i t i o n the enzyme within the complex. The analyses reported in t h i s thesis suggest that the i n t e r a c t i o n between RNA polymerase III and the sequences flanking the 5' boundary of the gene play a major role in determining the rate of complex u t i l i z a t i o n , that i s , Vo. This i n t e r a c t i o n might also e f f e c t the s i t e of t r a n s c r i p t i o n i n i t i a t i o n . I t i s conceivable that the 5' flanking sequences e f f e c t the formation of an "open promoter complex" (1) analogous to b a c t e r i a l RNA polymerase t r a n s c r i p t i o n systems thus influencing the rate of i n i t i a t i o n . Components within t h i s complex shown to recycle in the enzyme concentration curve reported here appear to include Factor IIIB and RNA polymerase III since nonactive p a r t i a l t r a n s c r i p t i o n complexes can be stimulated to transcribe by the addition of p u r i f i e d active t r a n s c r i p t i o n complexes (Loverne Duncan, unpublished r e s u l t s ) . Stage 4 I have assumed that Factor IIIB d i s s o c i a t e s from the complex either af t e r the formation of the t r a n s c r i p t i o n complex or shortly a f t e r i n i t i a t i o n of t r a n s c r i p t i o n . Factor IIIB might recycle l a t e r . Once t r a n s c r i p t i o n has i n i t i a t e d , elongation procedes r a p i d l y . 201 Stage 5 The elongation of RNA continues u n t i l RNA polymerase III reaches a termination signa l positioned 10 - 20 nucleotide downstream from the gene (73-75). 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