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Studies on the regulation of RNA polymerase in Escherichia coli Little, Robert 1980

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STUDIES ON THE REGULATION OF RNA POLYMERASE IN ESCHERICHIA COLI A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Microbiology Faculty of Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1980 (c) Robert Little, 1980 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements for an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e for reference and s tudy. I f u r the r agree t h a t pe rm i s s i on for e x t e n s i v e copy ing o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g ran ted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copy ing or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . Department o f ]f)lOCt1£  M  /  V The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date flpjJ. I 1 X 6 ) ABSTRACT The regulation of RNA polymerase subunit synthesis and its relationship to the expression of ribosome component genes have been investigated in strains of Escherichia coli having a mutation in one of the genes specifying either the 3 or 3* subunit of the core enzyme.' Particular attention has been focused on the L10 transcriptional unit (organization: P L l Q - rplJ(LlO) - rplL(L7/L12) - attenuator - rpoB(g) -rpoC(g') ). The mutant strain XH56 produces a defective 3' subunit which renders the RNA polymerase inactive in transcription initiation at 42°C; at somewhat lower temperatures, the RNA polymerase activity is only partially restricted. A temperature shift of this strain from 30°C to 39°C resulted in a rapid 5-fold increase in the transcription of the rpoB and CI genes and in the synthesis rates of the 3 and 3* subunits, indicating that 3 and 3' synthesis is regulated primarily at the transcriptional level. Transcription of the a subunit gene, located in the spc-str region of the chromosome, was also enhanced. Transcription of the lacZ gene (coding for 3-galactosidase) was decreased to undetectable levels, indicating that the dramatic increase of rpoB and C. transcription occurred at the expense of transcription of other operons. The mutant strains Ts4 and A2R7 produce defective 3' and 3 subunits respectively which are unable to assemble into core RNA polymerase at the nonpermissive temperature. In these strains RNA polymerase assembled prior to a temperature shift from 30°C to 42°C retains its activity but little or no enzyme is assembled after the shift. Prolonged incubation of these strains after such a shift produced a gradual 1.5- to 2-fold increase in the transcription of the rpoB and _C genes and in the synthesis rates of the 3 and 3' subunits. During the restrictions, transcription of ribosome component genes was essentially unchanged. RNA polymerase assembly was also inhibited in strains carrying both a temperature-sensitive amber suppressor mutation and an amber mutation in the rpoB gene. Under permissive conditions these amber mutations are suppressed by insertion of serine into the 3 protein at the UAG codon. After a temperature shift to 42°C, core RNA polymerase synthesis is restricted due to the failure to produce 3 in the non-polar amber strain MX515 and both 3 and 3' in the polar amber strain MX550. Core enzyme synthesized prior to the shift retains its activity. Inhibition of core enzyme synthesis in this manner resulted in a gradual stimulation of rpoB and C transcription; in the polar strain this was accompanied by a concomitant increase in the synthesis rate of the 3' subunit protein. The increase of rpoB and J] transcription involved both increased initiation at E^q and relaxed termination in the rplL-rpoB intergenic space. It was also observed that transcription of the a subunit gene was specifically stimulated during the restriction, suggesting that the regulatory mechanisms are specific for genetic units containing core RNA polymerase genes. These results therefore indicate that the mechanisms which govern the transcriptional frequency of operons containing RNA polymerase genes are coupled to the demand for active RNA polymerase; a sudden restriction of enzyme activity produces a rapid and dramatic increase of rpoB and JC transcription whereas a slow restriction results in only a gradual and less extensive induction. The regulatory mechanisms operating within the L10 transcription unit were accentuated by introducing the composite colEl plasmid pJC701 into the RNA polymerase activity mutant strain XH56.. All of the genes in the L10 transcription unit except the distally located rpoC are present on this plasmid and therefore were amplified in the transformed bacteria. The partial temperature inactivation of RNA polymerase activity in this strain allowed us to modulate the transcription of the proximal rplJ-rplL genes and the distal rpoB gene over a 10-fold and 30-fold range respectively. The observed imbalance in transcription between the proximal and distal portions of the L10 transcription unit strongly. suggest that the restriction has :two distinct effects: (i) it stimulates initiation at the major L10 promotor and (ii) it reduces termination at the attenuator located within or near the rplL-rpoB intergenic space. The synthesis rates of L7/L12 and 3 subunit proteins were also measured and compared to their respective mRNA levels under these conditions. The synthesis rate of L7/L12 protein and 3 protein varied by less than 2-fold and by 15-fold respectively. These measurements clearly indicate that translation of excess L7/L12 ribosomal protein mRNA is severely restricted and contributes to maintaining the balanced synthesis of ribosome components. The translational efficiency of 3 mRNA was also reduced by about 50%. Under the above conditions, 3 protein is produced in large excess relative to 3* subunit protein. Abstract ii Table of Contents v List of Tables List of Figures x Acknowledgements xi INTRODUCTION 1 I. Components of the _E. coli RNA polymerase . . . . 2 II. The transcriptional process 3 a. Initiation 3 b. Elongation 7 c. Termination 8 III. Regulation of RNA polymerase 10 a. Genetic organization of genes coding for RNA polymerase subunits . 10 b. The present investigation 13 MATERIALS AND METHODS 1 6 I. Abbreviations and chemicals 16 a. Abbreviations ^ b. Chemicals and radioisotopes 16 c. Media 17 d. Buffers and solutions 19 i. RNA preparations 19 ii. ADNA preparations 20 iii. Plasmid DNA preparations 20 iv. DNA-nitrocellulose filter preparation . . 21 v. SDS-polyacrylamide gel electrophoresis . . 21 vi. Urea-polyacrylamide gel electrophoresis . . 23 vii. Miscellaneous 23 II. Bacterial strains and growth conditions 24 III. Preparation of X  transducing phage DNA 27 IV. Preparation of plasmid DNAs 29 V. Pulse-labeled RNA preparation 30 VI. Preparation of DNA nitrocellulose filters . . . . 32 VII. DNA/RNA hybridization assays 35 VIII. Protein labeling and quantitation of 3 and 3' 37 IX. Quantitation of the relative synthesis rate of L7 and L12 39 a. Protein labeling of MX550 39 b. Protein labeling of XH56 and XH56-701 . . . . 40 c. Protein extraction and separation 40 d. Quantitation of the relative rates of L7/L12 synthesis 41 RESULTS PART A: Expression of RNA polymerase and ribosome component genes in mutants of Escherichia coli having conditionally defective RNA polymerases . ., . . 43 Summary 44 Introduction 45 Materials and Methods 47 Bacterial strains and growth conditions . . . . 47 Protein labeling and quantitation of 3 and 6' subunits of RNA polymerase 48 Nucleic acid preparation and hybridization . . . 48 Results 52 RNA synthesis in the RNA polymerase mutant strains 52 Synthesis of 3 and 3* subunits of RNA polymerase at restrictive temperatures 52 Transcription of rpoB and C and ribosomal protein genes in RNA polymerase assembly-defective mutants 56 RNA polymerase gene expression in a mutant defective in initiation of RNA chains . . 59 Discussion 66 Regulation of 3 and 3; subunit synthesis . . . 66 Expression of other genes during the restriction . 68 PART B: Regulation of RNA polymerase synthesis: Conditional lethal amber mutations in the 3 subunit gene . • 70 Summary 71 Introduction 72 Materials and Methods 75 Bacterial strains and growth conditions . . . . 75 Pulse labeling of proteins . . . . . . . . 75 Labeling of RNA and hybridizations 77 Results 78 Synthesis of 3 and 3' subunit proteins . . . 78 Transcription patterns during RNA polymerase limitation 82 Synthesis of L7/L12 ribosomal protein . . . 88 Discussion . . . . . . 93 PART C: Transcriptional and post-transcriptional control of ribosomal protein and RNA polymerase genes 96 Summary 97 Introduction 99 Materials and Methods 101 Bacterial strains and growth conditions . . . 101 Results 102 Genetics and regulation 102 Transcription with operons containing RNA polymerase genes 102 Synthesis of L7/L12, 3 and 3' protein . . . HO Discussion H6 LITERATURE CITED I 2 0 Table I Table II PART A: Table III PART B: Table IV Table V Table ..VI . PART C: Table VII Table VIII Table IX Bacterial strains 25 Structure of the DNA molecules used in DNA/RNA hybridization assays. 34 Relative rates of 3 and 3' subunit synthesis after a shift to restrictive conditions. 55 Quantitation of 3 and 3' subunit synthesis. 81 Transcription of RNA polymerase and ribosomal protein genes. 86 Quantitation of the synthesis rate of L7 and L12 protein. 92 Relative frequencies of transcription of ribosomal protein and RNA polymerase genes. 105 Relative synthesis rate of L7/L12 ribosomal protein. 113 Relative synthesis rate of RNA polymerase 3 and 3' subunits. H5 LIST OF FIGURES Figure 1 Figure 2 Part A: Figure 3 Figure 4 Figure 5 Figure 6 Part B: Figure 7 Figure 8 Figure 9 Figure 10 Part C: Figure 11 Figure 12 Figure 13 Genetic organization of the spc-str region of the IJ. coli chromosome. Structure of DNA probes used to determine transcription of the RNA polymerase 3 transcription unit.. Accumulation of cellular mass and RNA after a temperature shift to 42°C. Transcription of RNA polymerase and ribosomal protein genes in RNA polymerase assembly-defective mutants. Transcription of RNA polymerase, ribosome component and lacZ genes in a RNA polymerase activity-defective mutant. Relative transcription of rpoB and rpoC genes in a RNA polymerase activity-defective strain. Organization of the RNA polymerase cription unit. trans-Autoradiograms of pulse-labeled proteins separated on sodium dodecylsulfate poly-acrylamide gels. Hybridization of pulse-labeled RNAs to DNAs specifying ribosomal proteins and RNA poly-merase subunit proteins. Autoradiograms of pulse-labeled proteins separated on urea-polyacrylamide gels. Transcription of genes coding for RNA polymerase subunits and ribosomal proteins. Transcription termination at the attenuator following partial restriction of RNA polymerase activity. Autoradiograms of L7/L12 and 3 and 3' proteins separated by polyacrylamide gel electrophoresis. Page 12 33 53 58 62 64 73 80 84 91 107 109 ACKNOWLEDGEMENTS I would like to extend my sincerest appreciation to my advisor, Dr. Pat Dennis for his unwavering encouragement, invaluable conversations and general friendship during the course of this work. I also want to thank Dr. R.M. Blumenthal, Dr. G. Delcuve, W. Downing and H. Lewis for their support and fellowship. Thanks are also due to Rosemary Morgan for typing this thesis. Finally, I wish to thank the members of my thesis committee, Dr. J.J.R. Campbell, Dr. R.C. Miller, Dr. G. Tener and Dr. R.A.J. Warren for their constructive criticisms. INTRODUCTION The process of gene expression involves a complex sequence of events by which information encoded by the chromosome is rendered into a biologically active function. Overall gene expression may in fact be divided into two distinct events, transcription and translation. Transcription, the process by which specific sequences of DNA are transcribed into RNA inter-mediates, is mediated by RNA polymerase. The RNA products of transcription are subsequently utilized to direct the synthesis of specific proteins in the process of translation; the major components of the translation apparatus are ribosomes and transfer RNAs. In the bacterium Escherichia coli the regulation of gene expression occurs mainly at the transcriptional level. A knowledge of the regulatory events which serve to control the synthesis and activity of the transcriptional components, in t particular the RNA polymerase, is therefore of obvious importance. I Components of the E. coll RNA polymerase. In IS. coll a single RNA polymerase enzyme is responsible for all cellular transcription. The RNA polymerase core enzyme is a heteromultimer having the subunit structure C^BB' ( 8, 14, 145 ). The aggregate molecular weight of the core enzyme is approximately 400,000 to which each a subunit contributes 40,000, the 3 subunit about 155,000, and the 3' subunit about 165,000. In vitro, the core enzyme is able to synthesize random RNA transcripts from a DNA template, but the discrete selection of specific transcriptional start sequences is dependent upon an additional factor, the O sub-unit, having a molecular weight of about 90,000 ( 14, 15 ). When the a subunit is bound to the core enzyme the resulting a ' c r complex is termed the holoenzyme which has an aggregate molecular weight of about 490,000. This size appears to be substantially greater than that required strictly for transcription since the much smaller induced RNA polymerases isolated from IS. coli infected with bacteriophages T3 or T7 (molecular weights of 107,000 and 109,000 respectively) or Pseudomonas infected with bacteriophage gh-1 (molecular weight of 98,000) are also competent in specific recognition of transcription start sequences in the respective phage DNAs and the subsequent synthesis of discrete RNA transcripts (7). The greater size of the 15. coli RNA polymerase may reflect the genetic complexity of the organism and the demand for inter-action with regulatory molecules that alter its transcription specificity (136). II The Transcriptional Process. The transcriptional process may be divided into an intricate sequence of reactions in which the RNA polymerase holoenzyme selects specific start sequences, initiates an RNA chain, elongates the chain, and terminates the nascent chain at specific stop sequences with subsequent release of the enzyme from the DNA template (16, 17). a. Initiation. The initiation of transcription at discrete start sequences is related to the binding of the a subunit to the core RNA polymerase complex to form the holoenzyme. Although a does not appear to bind tightly to the core enzyme complex (135), its interaction with the core components induces a conformational change in the RNA polymerase (141). 4 This conformational change increases by about 10 -fold the rate of dissociation of non-specific core RNA polymerase-DNA complexes and induces the formation of highly stable open promotor complexes at the specific start sites (56). Presumably.then, the binding of a to the core enzyme induces a conformational change which allows the enzyme to recognize its specific starting sites for transcription (43,79,80). To impart specificity to the initiation process, it would be expected that the discrete promotor sites utilized for initiating transcription would have common elements within their sequences. Many promotor regions have been identified by the binding and/or protection of specific sequences of DNA by RNA polymerase holoenzyme under initiation conditions in the absence of the ribonucleotide triphos-phates (48,117,122,124). From these sequences, several general state-ments may be made concerning promotor organization and RNA polymerase recognition: First, most of the promotor sequences are enriched in AT base pairs and have an asymmetry in the distribution of purine and pyrimidine residues between the sense and anti-sense strands. Second, the seven base pairs centered about nucleotide -9+ have, with minor variations, the Pribnow base sequence TATRATR where R represents a purine (113). Third, the sequences between nucleo-tide -27 and nucleotide -37 exhibit a striking homology which is referred to as the -35 recognition region (123). Finally, the sequences of promotors from operons having similar metabolic functions or related regulatory mechanism exhibit more sequence homology amongst themselves than with promotor sequences of operons having different metabolic functions or regulatory mechanisms. The DNA fragments isolated by the RNA polymerase protection technique include the Pribnow box sequence but do not extend into the -35 recognition region of the promotor. These fragments are unable to rebind holoenzyme when subsequently used in RNA polymerase binding assays (122,113). Furthermore, mutations which significantly affect the rate of transcription have been shown to alter the nucleotide sequence of either the Pribnow box or the -35 recognition region (33, 34, 47, 91, 98, 111). In fact, RNA polymerase binding to DNA fragments containing promotors is dependent upon the presence of the -35 recognition region ( 2, 93, 94 ). These results there-fore suggest that the specific recognition of promotors by RNA polymerase is mediated by the -35 recognition region while the Pribnow box specifies the actual initiation site for transcription. This presumably requires the migration of RNA polymerase from the -35 recognition region to the Pribnow box region. The enrichment + nucleotide +1 = start of transcription. of AT base pairs and asymmetric distribution of purines and pyrimidines may enable the RNA polymerase to achieve this migration rapidly. Mechanisms affecting the initiation of transcription are primarily responsible for regulation of gene expression. From the above discussion it is apparent that these mechanisms could potentially act directly on the RNA polymerase, therefore altering the promotor recognition activity of the enzyme, or on the promotor regions them-selves to inhibit or enhance RNA polymerase binding. Although no well documented evidence is available for direct involvement of RNA polymerase under normal conditions, circumstantial evidence for altered RNA polymerase specificity due to alterations of the enzyme itself has been presented. When the RNA polymerase from T4 infected Ii. coli is isolated, several phage coded polypeptides are found to be associated with the enzyme ( 86). In addition, the a subunit has been shown to undergo a modification, each a subunit having an adenosine diphosphoribose residue (49). The effect of these modifi-cations appears to be an alteration in the specificity of the RNA polymerase which allows the T4 genes to be expressed preferentially at the expense of host stable RNA synthesis ( 77, 106). Other support for direct RNA polymerase involvement comes from studies on strains having mutations in RNA polymerase genes. Mutants which produce defective cr subunit (137) have been shown to alter the expression of the lactose operon and other catabolite repressible systems. It therefore seems possible that the interaction of factors with RNA polymerase may alter the specificity of promotor recognition. Alternatively, factors which modulate expression of operons may interact with the promotor to inhibit (negative control) or enhance (positive control) RNA polymerase binding. On the basis of studies with the lactose operon, Jacob and Monod proposed a region for negative control distinct from the promotor at which a regulatory molecule could bind to prevent RNA polymerase binding (62). The so-called operator regions to which repressors bind in the lactose operon ( 33, 90 ) and the tryptophane operon (110) have been shown to overlap the start site region, thereby preventing RNA polymerase from initiating transcription. Positive control mechanisms involved in the catabolite repression system of trans-criptional regulation have been suggested to act in a region located about 60 bases prior to the transcription start site defined by the Pribnow box in the lactose operon (33,99,109,117), and the galactose operon (99). Apparently, the cAMP-CAPf complex may bind to this site and enhance RNA polymerase binding either by direct interaction with RNA polymerase (47) or by acting at a distance to destabilize the DNA and allow the formation of RNA polymerase-DNA initiation complexes (33). However, the same region of the arabinose BAD operon exhibits no obvious sequence homology with the -60 region of the lactose or galactose operons (R. Schleif and G. Wilcox, cited in ref. 117). Furthermore, the interaction of the cAMP-CAP complex with the galactose promotor extends beyond the -60 region and in fact overlaps the -35 recognition region (134). These results suggest that direct interaction * between cAMP-CAP and RNA polymerase may play an important role in transcription initiation. Obviously, various combinations of negative and positive control mechanisms + . CAP = cAMP activator protein. of RNA polymerase binding are possible which permit various modes of control. For example, the X phage repressor, when in moderate concentrations, acts negatively on the rightward promotor but at the same time acts positively on the leftward promotor for its own synthesis (93). At high concentrations, however, repressor acts negatively on both promotors. This modulation in its effect is dependent upon three contiguous binding sites for repressor in the rightward operator. At moderate concentrations, repressor binds to two of these sites and represses initiation at the rightward promotor. Only when the third site is occupied by repressor is leftward promotor binding of RNA polymerase repressed. Recently, evidence has been presented suggesting cooperativity in repressor binding to the operator sites in which direct protein-protein inter-actions play a part (69) in regulating the expression from the leftward and rightward promotors. b. Elongation. Subsequent to promotor binding, migration to the initiation site (during which the a subunit is released) and initiation of RNA synthesis, the core RNA polymerase complex continues to elongate the nascent RNA chain. From studies on the rate of mRNA synthesis, it would appear that the variation in the rate of synthesis is dependent upon the frequency of initiation of transcription, the rate of ribonucleotides incorporated per RNA polymerase molecule remaining relatively invariant over a wide range of growth conditions (12, 13, 27, 96, 97, 125, 126). The rate of rRNA synthesis is also determined by the rate of RNA polymerase initiation at the rDNA promotors. However, in this instance, the chain elongation rate is somewhat greater than for mRNA and may increase with increasing transcription frequency (27). In 12. coli, at least five rRNA operons are present on the genome. Measurements of RNA synthesis show that rRNA synthesis accounts for up to 60-70% of the total transcription (28, 46). Under optimal conditions, the initiation frequency of each of the rRNA operons would therefore be about 1 sec ^ (139), a value much greater than other operons. When the nucleotide sequences of the promotor regions of three of the rRNA operons was determined, two RNA polymerase binding sites about 100 nucleotides apart were apparent (22, 143). The presence of two RNA polymerase binding sites may account in part for the high initiation frequency of rRNA transcription. c. Termination. Upon encountering a terminator sequence in the DNA, RNA polymerase ceases to migrate along the DNA and further incor-poration of ribonucleotides into the RNA chain is terminated. Subsequently, the RNA chain is released from the RNA polymerase and the core enzyme complex dissociates from the DNA template. In vivo, transcription termination in many instances is dependent upon the termination factor p , having a molecular weight of about 50,000 (119). The p factor is thought to bind to a hairpin region of the transcript close to the halt site of the RNA poly-merase and catalyze the release of the RNA polymerase from the template; it also exhibits an RNA-dependent nucleoside triphos-phatase activity ( 87 ). The NTPase activity is required for the termination activity ( 45, 57 ) and both of these activities are dependent on the presence of an RNA transcript ( 21). It has therefore been proposed that.p may act by migrating along the RNA transcript in the 5' to 3' direction.(the energy required for its movement being generated by the NTPase activity),encountering an RNA polymerase molecule stalled at a terminator (see below) and catalyzing the termination reaction (1 ). The presence of an impeding molecule on the RNA transcript, such as a ribosome in the process of trans-lating the RNA transcript, would prevent p from encountering the RNA polymerase. Just as certain sequences within the promotor region are implicated in RNA polymerase binding, common features are evident in the terminator regions recognized by p. The 3'-OH terminal sequences of the terminated RNA products of transcription are characterized by a run of 6 to 8 uridine residues which are preceded by a GC rich region ( 47 ). Other features which are generally present include a region of dyad symmetry which potentially generates a stem-loop structure in the RNA transcript and a CAATCAA sequence in the DNA which is located in the region which would correspond to the stem-loop dyad symmetry region ( 75, 120). The GC rich region may act to stall the RNA polymerase, allowing \ p interaction, while the stem-loop structure has been proposed as a secondary structure recognition for the p factor. However, the question of whether p per se is responsible for termination, or merely enhances termination has recently been raised by the demonstration that mutants having a defective 3 subunit appear to negate the requirement for p in termination (52). Ill Regulation of RNA Polymerase Synthesis. From the above discussion, it is evident that a comprehensive knowledge of the regulation of cellular transcription requires, in addition to other things, an understanding of the mechanisms which regulate RNA polymerase synthesis. Important to this under-standing is the organization of the.genes that code for the RNA polymerase components. a. Organization of genes coding for RNA polymerase subunits. Although it might be expected that genes coding for the core RNA polymerase subunits would be organized into a single genetic unit, these genes are in fact found in two distinct regions of the _E. coli chromosome. The genes coding for a and p are located in regions distinct from those in which the core component genes are situated. In studies on ribosomal protein gene organization, a number of A-transducing phages were isolated that carried chromosomal DNA from the spc-str region, located at about 72 min on the chromosome ( 65, 66, 67 ). Analysis of the proteins expressed by these transducing phages in UV-irradiated bacteria showed that the gene coding for the a subunit, rpoA, was located in this region of the chromosome (63). When the organization of the chromosomal genes carried by the transducing phages was determined the rpoA gene was found to be situated in an operon which also contained the genes for ribosomal proteins S4, Sll, S13 and L17 (see Figure 1 , 64, 68, 104 ). From the analysis of a single site mutation affecting the a subunit, it was determined that only one copy of the rpoA gene is present in the genome and this gene is located in the spc-str region ( 42). Resistance to the antibiotics rifampicin and streptolidigin, inhibitors of transcription initiation and elongation respectively, have been shown to reside in the 3 subunit of RNA polymerase ( 55, 114). Genetic mapping of mutations conferring resistance to these antibioticsallowed the rpoB gene, coding for the 3 subunit, to be located at 88 min on the chromosome. It was later shown that the gene for the 3' subunit, rpoC and genes coding for ribosomal proteins LI, L7/L12, L10 and Lll were also located in this region ( 67, 81 83, 88, 140 ). It has now been determined that the rpoB and C^  genes belong to a common transcriptional unit and are cotranscribed with two of the adjacent ribosomal protein genes rplJ (L10) and rplL (L7/L12) (36,84,103,143• The genetic organization of the transcription unit encompassing rpoB and C^  is shown in Fig. 2(p.33). The gene coding for the a subunit, rpoD, is located in the dnaG region, located at 66 min on the chromosome (50,53,100,101 ). Although the gene for ribosomal protein S21 (rpsU) has been mapped in this same region (129), there is no indication for cotranscription of the rpoD and rpsU genes. The gene coding for p factor, rho, has been shown to be identical to the .suA locus ( 58, 115, 116 118 ) and is located in the ilv region at about 83 min on the chromosome. No ribosomal protein genes have been located in this region. XtrkA H ^ Xspcl K E^ t Xspc2 K A9 h H A16 h — H 1 2.2 1.2 27 |5 10 t|4*J | 9 | 20 i 8.5 U I  \fus2 ^ r 1 7 / -- ^  / / » » / » » / e.5 11.2 j II --1 EF-T» EF-G (S7 S.2)P„j$$^ i J 5 1 3 1 10 • I • 4.6 • 2 2 j ^ LI7 a S4 SII(SI3)pa ,(LI5 L301 S3 (LIB L6 S8 SI4 L3 L24 LI4 ) LI6 L29MS3 SI9 L22L2L23L4XL3 SIO i Figure 1: Genetic organization of the spc-str region of the Ii. coli chromosome. b. The present investigation. During normal growth of E_. coli, the molar ratios of the individual a, $ and 3' subunits compared to core RNA polymerase is about 4:1:1 (35). The demonstration that 6 and 6' are present in absolute stoichiometric amounts whereas a is present in excess of the amounts required suggest that the amount of core RNA poly-merase in the cell is limited by the amount of 3 and 3' synthesized. At the outset of the studies presented here it had not been demonstrated that the rpoB and C genes were cotranscribed with the adjacent rplJ and L genes. However, a coordinate regulation of these genes was suggested from detailed investigations of their transcriptional activities (24). Over a range of steady-state growth conditions the genes coding for the 3 and 3' subunits of RNA polymerase were transcribed at about one-fifth the frequency of the adjacent ribosomal protein genes. These results were con-sistent with the measurements showing that the ratio of ribosome particles to RNA polymerase molecules was also about 5:1 (20, 35, 61, ). These results,-therefore, suggested that the rpoB and C^  genes are cotranscribed with the adjacent ribosomal protein genes; an attenuator present in the rplL-rpoB intergenic region normally terminates about 80% of the transcription initiated from P L 1 Q (24). The studies presented in this thesis were undertaken to determine the mechanisms, particularly in the L10 transcription unit, which regulate the synthesis of the core components of the _E. coll RNA polymerase. Since the knowledge of RNA polymerase regulation was expanding during the course of these studies, an introduction preceding each section indicates the status of the problem at the time the results in that section were concluded. The approach used to determine these mechanisms was to analyze the physiological effects of mutations in the rpoB and rpoC genes on the expression of the genes for RNA polymerase subunits and ribosome components. These effects have been determined at both the trans-criptional level, by DNA/RNA hybridization, and at the translational level, by determining the synthesis rates of particular proteins separated by polyacrylamide gel electrophoresis. The results obtained indicate that transcription of operons containing genes coding for RNA polymerase core components is selectively stimulated by a restriction of the overall transcriptional capacity of the cell; operons containing only ribosome component genes are not affected. The increased transcription of RNA poly-merase operons is apparently mediated by an alteration in the cellular pool of active RNA polymerase molecules which allows specific recognition of these operons at the expense of others (such as the catabolite-repressible lactose operon). Although the mechanisms which allow specific recognition of the RNA polymerase operons remain obscure,, the results demonstrate that the trans-criptional frequency of the rpoB and C^  genes is modulated by controls exerted both at the promotor, P^io' anc* a t t*ie trans-cription attenuator located in the rplL-rpoB intergenic region. The variation in the transcriptional frequency of the rpoB and ^  genes was in most instances accompanied by a coordinate variation in the synthesis rates of the 3 and 3' subunits. These results indicate that the synthesis of the 3 and 3'- proteins is regulated primarily at the transcriptional level. However, under conditions which potentially could have resulted in a large excessive production of the 3 subunit, achieved by a plasmid-mediated amplification of the rpoB gene in a strain having an activity defective RNA polymerase, the translational efficiency of 3 mRNA was decreased by about 50%. This result presumably reflects the existence of a post-transcriptional mechanism for maintaining near equal production of the 3 and 3' subunits under conditions which dissociate their normal coordinate synthesis. Similarly, synthesis of L7/L12 protein is restricted by a post-transcriptional mechanism when the level of rplL mRNA is elevated because of increased transcription initiation at P^io" This mechanism is presumably important in maintaining the balanced production of ribosomal proteins under conditions which dissociate ribosome and RNA poly-merase synthesis. I. Abbreviations and Chemicals. a. Abbreviations: bis-acrylamide: N,N"-methylene-bisacrylamide bis-tris: |bis(2-hydroxyethyl)imino-tris(hydroxy-methyl)methane DNA: deoxyribonucleic acid DNase: deoxyribonuclease EDTA: ethylene diamine tetraacetic acid IPTG: isopropyl-3-D-thiogalactoside MES: (2|N-morpholino|ethane sulfonic acid) MOPS: morpholinopropane sulfonic acid POPOP: p-bis[2-(5-phenyloxazolyl)|-benzene PPO: 2,5-diphenyloxazole RNA: ribonucleic acid RNase: ribonuclease SDS: sodium dodecyl sulfate SSC: standard saline citrate TEMED: N,N,N',N'-tetramethylethylenediamine TCA: trichloroacetic acid Tris: tris(hydroxymethyl)aminomethane b. Chemicals and radioisotopes: Acrylamide, bis-acrylamide and TEMED were obtained from Eastman Corp. Bis-tris, MES, MOPS, Tricine, RNase A, ampicillin, tetracycline and chloramphenicol were obtained from Sigma. RNase-free DNase I was obtained from Worthington Biochemicals. Rifampicin was obtained from Calbiochem. (3-mercaptoethanol was obtained from MCB. All radioactive isotopes, 3 3 14- 35 (5- H)uracil, L-(4,5- H)leucine, L-(U- C)leucine and L-( S)methionine were obtained from Amersham-Searle corporation. c. Media: MOPS minimal salts medium 5 x M per 1 final concentration (lx) MOPS 41.9 g 40 mM Tricine 3.59 g 4 mM NaCl 14.6 g 50 mM NH^Cl 1.44 g 5.4 mM Dissolve the ingredients in 750 ml dl^O. Adjust to pH 7.3-7.4 with KOH. Add dH20 to 1.0 1. concentrated (400 x) per 500 ml final concentration (lx) MgCl2.6H20 50.8 g 1 mM FeCl2 solution 10 ml 1.25 x 0 is made by diluting 1 ml of concentrated 0 in 400 ml dH20. 100 x ]?: per 100 ml final concentration (lx) K2HP04 2.21 g 1.27 mM 100 x S^: per 100 ml final concentration (lx) K„S0. 310 mg 218 yM I 4 FeCl2 solution per 100 ml final concentration conc. HC1 8 ml -FeCl2.4H20 5 g 10 pM CaCl2.2H20 184 mg 500 nM H3BO3 62 mg 400 nM MnCl2.4H20 40 mg 80 riM CoCl2.6H20 18 mg 30 nM CUC12.2H20 4 mg 10 nM ZnCl2 3 mg 10 nM (NH4)6MO7024.4H20 93 mg 3 nM ilete MOPS minimal salts medium is prepared with 20 ml 5 x M 80 ml 1.25 x 0, 1 ml 100 x P, and 1 ml 100 x S. M9 minimal salts medium: per 1 NH.C1 1.0 g 4 Na2HP04 7.0 g KH2P04 3.0 g NaCl 0.5 g After autoclaving, add 1 ml of a sterile 0.2 g/ml solution of MgS04. LB broth: per 1 Tryptone (Difco) 10 g Yeast extract (Difco) 5 g NaCl 5 g 1M NaOH 2 ml d. Buffers and Solutions: i) RNA preparations Medium C: NH.C1 4 Na HPO. 2 4 KH2PO4 NaCl SDS - lysis mixture: NaCl SDS EDTA 10 x TAE: Tris Na.azide EDTA - to pH 8.1 with HCl 10 x TMA: Tris NaN3 MgS04 - to pH 8.1 with HCl per 1 final concentration 2 g 40 mM 6 g 40 mM 3 g 20 mM 3 g 50 mM per 100 ml final concentration 0.58 g 0.1 M 0.50 g 0.5 % 0.37 g 0.01 M per 500 ml final concentration (xl) 60.05 g 0.1 M 3.25 g 0.01 M 168 mg 0.001 M per 500 ml final concentration (xl) 60.05 g 0.1 M 3.25 g 0.01 M 6.2 g 0.05 M 10 x SSC: NaCl Na^Citrate per 2 1 175 g 88 g final concentration (lx) 0.15 M 0.015 M ii) XDNA preparations Buffer B: 2M Tris, pH 7.5 1M MgS04 5M NaCl per 1 5 ml 5 ml 10 ml final concentration 10 mM 5 mM 50 mM 5 x NaCl-phosphate buffer per 400 ml Nal^PO^.H 0 13.8 g Na„HP0. 14.2 g 2 4 NaCl 11.1 g final concentration (lx) 100 mM 100 mM 100 mM - pH 7.1 iii) Plasmid DNA preparations Solution A: per 100 ml sucrose 25 g 1M Tris, pH 8.0 5 ml final concentration 25 % 0.05 M Lysis solution: 1M Tris, pH 8.0 0.5M EDTA, pH 8.0 Triton X-100 per 100 ml 5 ml 12.4 ml 2 ml final concentration 0.05 M 0.062 M TEN buffer: IM Tris, pH 8.0 0.5M EDTA, pH 8.0 5M NaCl TE buffer: IM Tris, pH 8.0 0.5M EDTA pH 8.0 iv) DNA-nitrocellulose Solution X: 1M HCl IM Tris, pH 8.0 20 x SSC + 30 mM MgCl2 Na.citrate NaCl MgCl2 v) SDS Polyacrylamide SDS-lysis mixture: 1.5M Tris, pH 6.8 10% SDS glycerol 3-merc ap toe thano1 0.1% bromphenol blue per 1 final concentration 10 ml 10 mM 2 ml 1 mM 2 ml 10 mM per 1 final concentration 5 ml 5 mM 2 ml 1 mM filter preparation per 1 final concentration 100 ml 0.1 M 50 ml 0.05 M per 1 final concentration (lx) 88 g 15 mM 175 g 150 mM 60.9 g 30 mM gel electrophoresis per 100 ml 5 ml 20 ml 10 ml 5 ml 1 ml final concentration 0.0625 M 2 % 10 % 5 % 0.001 % 7.5% SDS-polyacrylamide slab gels 1.5M Tris, pH 8.8 10% SDS 30% acrylamide:0.8% bis-acrylamide dH20 per 40 ml final concentration - degas 1% TEMED 1% (NH4)2S208 10 ml 0.4 ml 10 ml 12.5 ml 4.0 ml 2.8 ml 0.375 M 0.1 % 7.5 % 0 . 1 0.07 '% Buffer overlay: 1.5 ml of 1.5 M Tris, pH 8.8 and 4.5 ml dH20 3% SDS-polyacrylamide stacking gel 1.0M Tris, pH 6.8 10% SDS 30% acrylamide:0.8% bis-acrylamide dH20 - degas 1% TEMED 1% (NH4)2S20g per 10 ml final concentration 1.25 ml 0.125 M 0.1 ml 0.1 % 1.0 ml 3.0 % 6.0 ml 1.0 ml 0.7 ml 0.1 0.07 % 10 x Running buffer: Tris glycine SDS - pH 8.3 per 1 30.9 g 144 g 10 g final concentration (lx) 0.025 M 0.192 M 0.1 % vi) Urea-Polyacrylamide gel electrophoresis 10 x upper buffer: per 500 ml final concentration (lx) Bis Tris 20.92 g 20 mM pH to 7.0 with about 44 ml of 8 g MES in 50 ml of H20 10 x lower buffer: per 500 ml final concentration (lx) Bis Tris 30.34 g 30 mM pH to 6.0 with about 7 ml of acetic acid 2 x gel buffer: per 50 ml final concentration (lx) 9M urea 50 ml 9 M Bis tris 1.19 g 10 mM pH to 5.0 with about 0.95 ml of acetic acid 4% urea-polyacrylamide gel: per 50 ml final concentration 9M urea 18.25 ml 3.3 % 30% acrylamide:0.8% bis-acrylamide 6.67 ml 4 % 2 x gel buffer 25 ml 1 x (gel buffer) TEMED 50 yi 0.01 % - degas 10% (NH4)2S20g 400 1 0.8 % vii) Miscellaneous Scintillation fluid: POPOP 0.1 g PPO 4.0 g Toluene 1.0 1 II. Bacterial Strains and Growth Conditions. The characteristics and source of the bacterial strains used in these studies are listed in Table I. The temperature sensitive mutations carried by the mutant strains TS4 and XH56 were generated by nitrosoguanidine co-mutagenesis and £ selecting for rif , temperature sensitive clones. The temperature-sensitive mutation in strain A2R7 was obtained using the rif° selection method following 2-aminopurine mutagenesis (18). Briefly, the rif° selection is a procedure by which inactivation of a temperature-sensitive, but rifampicin-sensitive,6 subunit at a high temperature r s s in a rif /rif heterodiploid enables an otherwise rif heterodiploid to grow in the presence of rifampicin (3 ). The isogenic wild type and mutant strains X240, TS4, A2R7 and XH56, were derived by PI trans-duction of the temperature sensitive mutations to 12. coli strain X239 (F~, his, metB, bfe, purD, argH2, rpsL, lac) (73). The wild-type strain X240 was obtained using a PI lysate from a wild-type + + donor and selecting Arg Pur transductants. The mutant strain A2R7, in addition to the temperature-sensitive rpoB2 mutation, also carries the spontaneous rifampicin resistant mutation rpoB7. Strain XH56-701 is identical to XH56 except that it also harbours the ColEl composite plasmid pJC701 (see Fig.2 ); the bacterial substitution on this plasmid contains the prox:imal portion of the L10 transcription unit including the promotor P^^qj rplJ, rplL, rpoB and the associated regulatory regions but does not carry the distally located rpoC gene ( 19,29). Cultures were grown in M9 minimal salts medium supplemented -3 with 0.2% glucose or 0.2% glycerol (and 10 M IPTG), L-methionine Table I: Bacterial Strains Strain Genetic Characteristics Source X240 F~, his, metB, thi, rpsL, lac J. Miller and J. Kirschbaum (18) TS4 F , his, metB, thi, rpsL, lac, rpoC4(ts) J. Miller and J. Kirschbaum (18) A2R7 F~, his, metB, thi, rpsL, lac, rpoB2(ts), J. Miller and rpoB7 J. Kirschbaum (18) XH56 F~, his, metB, thi, rpsL, lac, rpoC56(ts) J. Miller and J. Kirschbaum (18) XH56-701 F , his, metB, thi, rpsL, lac, rpoC56(ts)/ H. Lewis and MX515 MX550 pJC701 trp(am), leu(am), lacZ(am), galK(am), galE(am), tsx, strA, relA, SupD43,74, sueA, rpoB (Ci, non-polar amber) trp(am), leu(am), lacZ(am), galK(am), galE(am), tsx, strA, relA, SupD43,74, sueB, rpoB (38, polar amber) P.P. Dennis M.P. Oeschger (107) M.P. Oeschger (3-07) (50 yg/ml), L-histidine (50 yg/ml) and thiamine (0.5 yg/ml) at the permissive temperature of 30°C for at least 8 generations prior to experimentation. The mutant strains MX515 and MX550 are isogenic with the exception that MX515 has the rpoB (C i, non-polar amber) and sueA mutations and 14X515 has the rpoB (30, polar amber) and sueB mutations. The supD 43,74 mutation is a temperature sensitive suppressor mutation which inserts the amino acid serine at UAG amber termination codons (108). The sueA and sueB mutations enhance suppression by the supD,43,74 mutation (Oeschger, M.P., and J.T. Wiprudjand Oeschger, M.P.,; M.S. Oeschger, J.T. Wiprud and S.L. Woods, both submitted for publication). Cultures were grown in MOPS minimal salts medium supplemented with 0.2% glucose, a synthetic mixture of 16 natural amino acids (minus cys, met, tyr, phe) (102) and thiamine (1 yg/ml) at 25°C for a minimum of 20 hr prior to experimentation. Under these conditions, the doubling times of the cultures were consistently 150-180 mins. Cellular mass accumulation was monitored as the absorbance at 460 nm (A^q) . Temperature-shifts of cultures were imposed at an A460 0*25 - 0.40. In all cases, experimental measurements were achieved when cultures had an A460 less than 1.0. Under the con-ditions used, sufficient nutrients were included in the media to allow exponential phase growth at the permissive temperature to an A460 °f at least 3.0. Cellular RNA accumulation was monitored as the t*ie acid-insoluble, alkali-labile material. Samples of 2 ml. of culture were precipitated in 0.2 ml of 100% TCA (final concentration of 10%) on ice for 20 min. The precipitates were filtered onto 2.4 cm glass fibre filters . (grade 984H, Reeve Angel) which were then placed into 1.5 ml of 0.2N NaOH and stored at 20°C for 16 hr. Perchloric acid (1.5 ml of a 1/15 dilution of concentrated perchloric acid) was added and precipitation of acid-insoluble material was accomplished by storing on ice for at least 1 hr. Aliquots were filtered through 2.4 cm nitrocellulose (0.45 ym, Millipore) and the A£gQ filtrate was determined. III. Preparation of X  Transducing Phage DNA. Bacterial strains lysogenized with X  transducing phages and ACI857 helper phage were inoculated into 2 1 of LB broth and grown at 30°C for 16 hr. The overnight cultures were then used to inoculate 20 1 fermenter batches of LB broth which had been equilibrated to 30°C. The cells were grown to an A^q of about 1.0 and the temperature was then increased to 42-44°C for 30 min. The temperature was reduced to 37°C and incubation was continued for 3 hr. The cells were harvested in a Sharpies continuous flow centrifuge and then suspended in 100 ml of Buffer B. The cells were lysed by incubation at 37°C for 10 min> with gentle mixing in the presence of 20 ml of CHCl^. Following the addition of 200 yg of DNase (final concentration of 1 yg/ml) incubation was continued a further 15 min. The suspensions were then stored at 4°C for 16 hr. The phage particles were separated from the bulk of the cellular debris by centrifugation at 10,000 xg for 30 min. Up to 25 ml of the supernatant was then loaded onto block gradients which had been prepared in 38.5 ml capacity nitrocellulose tubes and composed of, from bottom to top, 3 ml of CsCl of density 1.7 g/ml, 2 ml of CsCl of density 1.5 g/ml, 3 ml of CsCl of density 1.3 g/ml, and 4 ml of 20% sucrose. The CsCl and sucrose solutions were made using Buffer B. The gradients were centrifuged in an SW27 swinging bucket rotor (22,000 rpm for 2 hr at 5°C). The band corresponding to the position of phage particles (approximate density of 1.5 g/ml) was collected by carefully inserting a syringe through the top of the gradient until the needle was just below the phage band and withdrawing the phage. The suspensions were adjusted to a CsCl density of 1.5 g/ml and transferred to 13.5 ml capacity nitrocellulose tubes, a maximum of 7.5 ml per tube. The gradients were overlayed with mineral oil, capped, and centrifuged to equilibrium in a 50 Ti rotor (25,000 rpm for 22 hr at 5°C). The band corresponding to A transducing phage particles was removed by syringe through the sides of the tubes and dialyzed against NaCl-phosphate buffer for 18-20 hr. To obtain the DNA, the transducing phage particles were disrupted by treating 1.5 ml of phage suspension, 3.5 ml of NaCl-phosphate buffer, and 0.15 ml of 10% SDS (final concentration of 0.33%) with 5 ml of phenol saturated with NaCl-phosphate buffer. The mixtures were shaken gently for 30 sec and the aqueous and phenol phases were separated by centrifugation at 7,500 x g for 4 min. The aqueous phase was removed and extracted three more times with phenol saturated with NaCl-phosphate buffer. The aqueous phase was then dialyzed for 48 hr against 10 mM EDTA, pH 8.1, followed by dialysis against 10 mM Tris-acetate, pH 8.1 for 24 hr. The DNA solutions were stored at 4°C in the presence of chloroform. IV. Preparation of Plasmid DNA. Strains bearing plasmids were grown in M9 minimal salts medium supplemented with 0.4% glucose, 0.04% Casamino acids, tryptophan (50 yg/ml), thiamine' (50 yg/ml) and uridine (50 yg/ml).Overnight cultures,which had been grown in the same medium but in addition contained antibiotics which were selective for plasmid harbouring bacteria,were inoculated into fresh media (3-6 1) at a dilution of 1:50. The antibiotics used in the overnight culture medium were rifampicin (50 yg/ml) for growing pJC701, pJC703 and pJC720, ampicillin (100 yg/ml) for growing pSP 11, and tetracycline (10 yg/ml) for growing pNF 1564. The cultures were grown to an A46O 1.5-1.8 and chloramphenicol (80 mg/ml in 95% ethanol) was added to a final concentration of 200 yg/ml followed by continued incubation for 16-18 hr. The cells were harvested by centrifugation in a Sorvall GS-3 rotor at 7,400 xg for 10 min at 4°C. The cells were resuspended such that the cells from 1 L of medium were suspended in 10 ml of solution A and transferred to 40 ml capacity polycarbonate OakRidge-type centrifuge tubes. To each 10 mis of resuspended cells, 2 ml of 0.5M EDTA, pH 8.0 and 2 ml of a 4 mg/ml solution of lysozyme in solution A (final concentration of about 500 yg/ml) were added and the suspensions were mixed and incubated on ice for 15 min. Lysis solution was then added at a volume of 14 ml per 10 ml of resuspended cells, mixed in quickly, and lysis was achieved by incubation on ice for 10 min. Cleared lysates were obtained by centrifugation of the suspensions at 48,000 xg for 1 hr in a Sorvall SS-34 rotor. Per 5 ml of cleared lysate, about 4.9 g of CsCl were added to obtain a final density of about 1.776 g/ml (Refractive index ri „=!•4062). The cleared lysates were then distributed into 38.5 ml capacity nitrocellulose tubes to a maximum of 30 ml and overlayed with 0.5 ml of a 2 mg/ml solution of ethidium bromide per 7 ml of cleared lysate solution. In all subsequent steps in which DNA was in contact with ethidium bromide, minimum light was used to minimize any nicking of the DNA. The tubes were filled with mineral oil, capped and then inverted several times just prior to placing into the centrifuge to mix the cleared 'lysates and ethidium bromide phases. The samples were centrifuged to equilibrium in a 60Ti rotor (43,000 rpm for 44 hr at 15°C). The bands corresponding to covalently closed circular plasmid DNA were collected by puncturing the tubes through the side with a 21 gauge needle and removing the DNA with a syringe. The extracted bands were placed into 13.5 ml capacity nitrocellulose tubes and recentrifuged to equilibrium (36,000 rpm in a 50 Ti rotor for 48 hr at 15°C). The bands corresponding to plasmid DNA were collected as described above and the ethidium bromide was removed by extraction with CsCl-saturated isobutanol. The DNA was then dialyzed at 4°C for 24 hr through several changes of TEN buffer or TE buffer. The volume and &-26Q dialyzed DNA preparations were determined and, using a conversion factor of 20 A26o/ml being equivalent to 1 mg/ml of DNA, the yield of each preparation was deter-mined. Typically, 1 L of culture yielded in the range of 3-5 mg of plasmid DNA. The DNA preparations were checked for purity by restriction enzyme analysis. V. Pulse-Labelled RNA Preparation. Radioactive labelling and processing of RNA was carried out using the method of Dennis and Nomura (31). Samples (10-20 ml) of cultures 3 were pulse-labelled for 1.0 min with (5- H)uracil ( 25 Ci/mmol; 10 yCi/ml). Incorporation of radioactive uracil was terminated by pouring the cells on ice which had been cooled to -70°C in the presence of Na azide (final -3 concentration 10 M). The cells were harvested by centrifugation at 10,000 xg for 5 min at 0°C and resuspended in 1 ml of medium C and 10 ^ M Na azide. The cells were lysed by the addition to 1 ml of SDS-lysis mixture at 95 C for 20 sec. The samples were then extracted three times with 2 ml of TAE-saturated phenol. The phenol phases were pooled and reextracted with 2 ml of TAE. The aqueous phases were combined (4 ml) and the NaCl concentration was adjusted to 0.2 M by the addition of 0.25 ml of 4 M NaCl. The nucleic acids were precipitated by the addition of 8 ml of 95% ethanol and stored at -20°C for 18 hr. The precipitate was collected by centrifugation at 10,000 xg for 20 min at -10 C and resuspended in 2 ml of TAE. Residual phenol was removed by five extractions with 2 ml of ether. Residual ether was removed by passing +2 N 2 gas over the sample. The Mg concentration was adjusted to 5 mM by the addition of 0.2 ml of 50 mM Mg.SO^, 10 yl of a 1 mg/ml solution of RNase-free DNase in TMA was added (final concentration 5 yg/ml), and the samples were incubated at 20 C for 20 min to digest the DNA. The EDTA concentration was adjusted to 5 mM by the addition of 0.25 ml of 50 mM EDTA and the samples were extracted two times with 2.5 ml of TAE-saturated phenol. The phenol phases were pooled and reextracted with 1.5 ml of TAE buffer. The NaCl concentration of the combined aqueous phases (4 ml) was adjusted to 0.2 M and the RNA was recovered after precipitation in 95% ethanol as described above. The RNA was dissolved in 2 ml of 2 x SSC and residual phenol removed by ether extraction (see above) . The t*16 preparations were determined and the concentrations were adjusted to 50 yg of RNA per ml in 2 x SSC. An A26O is equivalent to 50 yg per ml of RNA. Samples of the preparations (10-20 yl) were precipitated at 0°C for 20 min in 2 ml of 5% TCA with 20 yl of stationary phase carrier cells. The precipitates were collected by filtration onto nitrocellulose filters (0.45 ym pore; Millipore) which were then dried and the amount of radioactivity determined by counting in 5 ml of scintillation fluid. VI. Preparation of DNA-Nitrocellulose Filters. The structure of the DNA probes used in hybridization assays are shown in Figures 1 and 2 and Table II . For DNA probes having ^ molecular weights greater than 8.0 x 10 , 3.0 x 10 moles of DNA (equivalent to about 5 yg/ml of Xdspc 1 DNA) were diluted in 90 mis of 2 x SSC, assuming 20 A26o/ml i s equivalent to 1 mg/ml of DNA. The DNAs were denatured by the addition of 5 ml • of 6M NaOH (final concen-tration of 0.33 M) and incubated at 20°C for 1 hr. The solutions were neutralized by the addition of 5 ml of 6M HCl and immediately immobilized 2 by filtration onto 10,400 mm nitrocellulose membrane filters (0.45 ym pore-size; Millipore Corp.), which had been presoaked and rinsed with 2 x SSC, at a flow rate of about 50 ml/min. The filters were washed with 2 x 100 ml of 2 x SSC, and then air dried for 1 hr. The DNA was baked onto the filters at 80°C for 3 hr in an evacuated dessicator. For the DNA probes having molecular weights less than 8.0 x 10 , the DNA (1.67 x 10 _ 1 1 moles) was diluted into 20 ml of 2 x SSC and 0.34 ml of 6 M NaOH (final concentration of 0.1M)was tpoB rplLrplJ rplA rplK pJC 703 701 | m lA- ///. pJC 720 XDNA 0 Col Ei DNA pNF 1564 Figure 2: Structure of the DNA probes used to determine transcription of the RNA polymerase 3 transcription unit. Table II Structure of the DNA molecules used in DNA/RNA hybridization assays. DNA probe Chromosomal genes Molecular weight Molecular weight of chromosomal of probe polypeptides specified by probe X - 30.0 X 106 -Xdtrk aroE, trkA 29.0 X 106 -Xdspcl aroE, rplE, trkA, F, N; rpsD, E, F, H, K, M, Q; rpmD, rpoA 29.0 X io6 300,000 Xilv5 rrnC 29.0 X 106 -Xplac lacZ 29.0 X 106 120,000 pJC701 rplK, A, J, L; rpoB . 8.8 X 106 274,000 pJC703 rplK, A, J, L; rpoB, rpoC 17.5 X 106 394,000 pJC720 rpoB, rpoC (COOH-terminus of rplL) 16.0 X 106 327,000 PNF1564 COOH-terminal rplJ - NH^-terminal rplL 2.4 X io6 23,000 pSPll internal portion of rpoA 3.1 X 106 25,000 added. The solution was heated at 67°C for 10 min. Following the addition of 20 ml of solution X, the DNA was heated at 100°C for 5 min, and then cooled rapidly by the addition of 140 ml ice-cold dH20 and 20 ml of 20 x SSC + 30 mM MgCl . The DNA was filtered onto nitrocellulose membrane filters (0.45 ym; Sartorius) and processed as described above. For use in hybridization assays, the individual filters were cut 2 using a number 5 cork-bore into circles of 57 mm . Each filter therefore contained about 167 fmoles of DNA (equivalent to 5 yg/filter of Adspcl DNA) . VII. DNA/RNA Hybridization Assays. The transcription of specific chromosomal regions was monitored by hybridizing the pulse-labeled RNA preparations to the specific DNA probes immobilized on nitrocellulose filters. The determinations were achieved by using 50, 100, 150 and 200 yl inputs, equivalent to 50 to 200 yg, of RNA for each hybridization series. Separate hybridizations were carried out in determining transcription of adjacent or overlapping regions of DNA contained on different DNA probes. In all cases, the separate hybridization series included blank (no DNA) and appropriate control filters. The hybridization assays were incubated for 18 hr at 67°C in a final volume of 2 ml of 2 x SSC. The filters for each RNA preparation were removed and washed three times with 20 ml of 2 x SSC. Following the addition of 5 ml of 2 x SSC, 20 yl of a 1 mg/ml solution of RNase A in 2 x SSC were added. The RNase stock solution had been boiled for 10 min to destroy any contaminating DNase activity. The final concentration of RNase A was approximately 2 yg/ml. The filters were incubated in the presence of RNase A for 40 min at 20°C. The filters were then washed four times with 20 ml of 2 x SSC and dried. The amount of radioactivity bound to each filter was determined by counting in 5 ml of scintillation fluid in a scintillation counter. The A and Adtrk DNAs were employed to measure non-specific hybrid-ization. The Adtrk DNA contains the aroE and trkA genes located near 72 min on the E.coli chromosome (see Fig. 1 ). The Ad spcl DNA, in addition to aroE and trkA, contains a cluster of genes coding for 15 ribosomal proteins and the a subunit of RNA polymerase which are also located in this region of the chromosome ( 63^66 ). The difference in the amount of radioactivity hybridized to Adspcl and Adtrk DNAs there-fore represents that mRNA which is homologous to the DNA of the ribosomal protein gene cluster and the rpoA gene. The pSPll DNA contains a Hind III fragment which specifies a portion of the RNA polymerase a subunit and hybridizes exclusively a.mRNA (S. Pederson, personal communication). The composite plasmid pJC720 carries chromosomal DNA which specifies the COOH terminal portion of ribosomal protein L7/L12 and the entire 3 and 3' RNA polymerase subunits (see Fig 2 ; 19 ). This DNA therefore hybridizes predominantly 33' mRNA ( 24). The pJC703 composite plasmid, in addition to the rpoB and £ genes, carries the DNA sequences for the rplK,A,J and L. genes which specify. ribosomal proteins Lll, Ll, L10 and L7/L12 respectively. Therefore, the difference in hybridization to pJC703 and pJC720 DNAs represents the RNA which is homologous to the DNA of the ribosomal protein gene cluster adjacent to rpoB and The pNF 1564 DNA contains a 658 nucleotide Eco.Rl-Pst restriction enzyme fragment which extends from the COOH terminal portion of rplJ to the NH3 terminal portion of rplL (29) and hybridizes mRNA specific for this region. The pJC701 DNA contains the rpoB gene.and the adjacent ribosomal protein genes (see Fig. 2 ; 19 ). When used in competition experiments with pJC720 DNA, the pJC701 DNA competes for the RNA which is homologous to rpoB DNA but not rpoC DNA. Therefore, the reduction of radioactivity hybridized to pJC720 DNA in the presence of excess pJC701 DNA is rpoB mRNA, the residual radioactivity hybridized to pJC720 DNA is rpoC mRNA. For competition hybridizations, constant amounts of RNA preparations were hybridized in a series of vials each containing 1 A DNA filter, 2 pJC720 DNA filters and having 0, 1, 2, 3, 4, 6 or 8 pJC701 DNA filters in a final volume of 2 ml of 2 x SSC (for a discussion of competition hybridizations, see 24 ). The A ilv5 DNA contains a rRNA transcription unit and hybridizes almost exclusively stable rRNA ( 19,24 ). The A plac5 DNA carries the lac Z gene and hybridizes mRNA which is transcribed from the lactose operon DNA. For the pSPll, pJC720, pJC703, pNF1564, A ilv5 and Aj"Plac5 DNA filters, non-specific binding of radioactivity in the hybridization assays was corrected for by subtracting the amount of radioactivity associated with A DNA filters. VIII.Protein Labelling and Quantitation of g and 3'. Cellular proteins were radioactively labelled under permissive conditions or. at various times following temperature shifts to non-permissive or semi-permissive conditions by the addition of L-(U"'"^ C) 35 leucine (specific activity 342-348 mCi/mmol; 2 liCi/ml) or ( S) methionine (specific activity 608 Ci/mmol; 2 yCi/ml) to 2.5 or 5.0 ml volumes of cultures. Incorporation was terminated after 30 min by the addition of excess non-radioactive leucine (50 yg/ml), leucine (50 yg/ml) and isoleucine (50 yg/ml), or methionine (50 yg/ml). Samples were incubated a further 2.5 min to allow for completion of nascent polypeptide chains. The cells were harvested by centrifugation at 10,000 x g for 5 min, resuspended in 250 or 500 yl of SDS lysis mix, and disrupted by boiling for 2 min. Cellular proteins were separated by electrophoresis in the presence of SDS on 1.5 mm thick 14 cm long 7.5% polyacrylamide slab gels with 3 cm long 3% polyacrylamide stacking gels ( 76,;127). Up to 25 yl- of each sample were loaded per well, (4 mm wide x 1.25 cm long) and electrophoresis was achieved by first stacking the proteins at 7.5 mA for 1 hr followed by applying a 25 mA current for 7-8 hr. Following electrophoretic separation, the g and g' subunits had migrated 6-7 cm into the gel and were separated from each other by about 3 mm. The gels were stained for 15 min with 0.1% Coomassie blue in 50% TCA and then destained in 7% acetic acid for 18 hr. The gels were dried and autoradiograms were obtained using Kodak XR-2 X-ray film. The relative rate of g and g' subunit synthesis was determined by two different methods. In the first, the bands on autoradiograms which corresponded to the g and g* subunits were scanned with a Quick-Scan Jr scanner (Helena Institute, Beaumont, Tex.) equipped with an integrator. The areas of the g and g' peaks were determined, corrected for differences in the amounts of total radioactivity applied to the gels as evaluated by scintillation counting of 5% TCA precipitations of 5-10 yl aliquots of the protein extracts, and compared. In the second method, the radioactive bands corresponding to the g and g' proteins were cut from the gels, oxidized with 0.3 ml of 40% E^O at 37°C for 16 hr, treated three times with 0.25 ml of catalase '(4 mg/ml in 10 mM tris, pH 7.4) to inactivate residual I^ O;?, an(* t*ie amount of radioactivity in the bands was determined directly by scintillation counting in 20 ml of 3a70B preblended liquid scintillation cocktail (Research Products International Corp.). The amount of radioactivity in the 6 and 3' bands was then corrected for differences in the total amount of radioactivity applied to "the gels. IX. Quantitation of the Relative Synthesis Rate of L7 and L12. a. Protein labelling of MX550. Samples of 5 ml from a culture of MX550 growing at 25°C or at 90 min following a temperature shift to 43°C were pulse-labelled for 5 35 min with ( S)methionine (specific activity 608 Ci/mmol; 2 yCi/ml). The samples were incubated a further 2.5 min in the presence of excess non-radioactive methionine (50 yg/ml) to allow for completion of nascent polypeptide chains. The cells were harvested by centrifugation at 10,000 xg for 5 min. Carrier cells were prepared by incubating 50 ml of an exponential phase culture of MX550 growing at 25°C with 3 L-(4,5- H) leucine (specific activity 342 mCi/mmol; 2 yCi/ml) for one generation. In this case, non-radioactive leucine was omitted from the growth medium to increase the labelling efficiency. The culture was divided into 25 ml aliquots and the cells were harvested by centrifugation at 10,000 xg for 5 min. b. Protein labelling of XH56 and XH56/701. Samples of cultures (5 ml) growing at 30°C or at various times following a temperature shift to 38.5 C were exposed to L—(4,5— H) leucine (specific activity 53 Ci/mmol; 20 yCi/ml) for 5 min. Incorporation of radioactive leucine was terminated by the addition of excess non-radioactive leucine (50 yg/ml) and isoleucine (50 yg/ml) and the samples were incubated a further 2.5 min. to allow for completion of nascent polypeptide chains. The cells were harvested by centrifugation at 10,000 xg for 5 min. Carrier cells were prepared by labelling 150 ml 14 of an exponential phase culture of XH56 growing at 30°C with L-(U- C) leucine (specific activity 342 mCi/mmol; 2 yCi/ml) for two generations. The culture was divided into 25 ml aliquots and the cells were harvested by centrifugation at 10,000 xg for 5 min. c. Protein extraction and separation. Carrier cells were resuspended in 5 ml of 0.05 M tris, 0.01 M EDTA, pH 8.1 and mixed with the pulse labelled cells. The mixed cells were harvested by centrifugation and resuspended in 0.3 ml of 0.05 M tris, 0.01 M EDTA, pH 8.1. The cells were disrupted by three cycles of freezing and thawing in the presence of 10 yg of lysozyme (10 yl of a 1 mg/ml solution of lysozyme in 0.05 M tris, 0.01 M EDTA, pH 8.1) +2 followed by two 10 sec sonication treatments. The Mg concentration was adjusted to 0.2 M by the addition of 60 yl of a IM solution of Mg acetate and the proteins were extracted with 0.72 ml of glacial acetic acid at 0°C for 2 hr. The acid soluble fractions following centri-fugation at 10,000 xg for 10 min were dialyzed for 18 hr against 120 ml of 8 M urea, 0.05 M 3-mercaptoethanol (100 mis 9 M urea, 90 pi 3-mercaptoethanol, and 20 mis dl^O). The volumes of the extracts were measured and lOx upper buffer was added to achieve a final concentration of lx upper buffer, and bromphenol blue was added to 0.1%. The acid soluble proteins were separated by polyacrylamide gel electrophor esis in the presence of 8 M urea ( 78)• These gels were 14 cm x 17 cm x 1.5 mm and had a final polyacrylamide concentration of 4%. The upper buffer electrode was the anode and the lower buffer electrode was the cathode. Samples of the protein extracts were applied to the wells which had been rinsed thoroughly and the proteins were separated by electrophoresis without cooling at 40 mA for 6-7 hr with continuous circulation of the upper buffer with an external reservoir. The gels were fixed with IN HCl for 20 min, rinsed with 7% acetic acid for 15 min. and dried. d. Quantitation of the relative rates of L7/L12 synthesis. Autoradiograms of the dried gels were obtained using Kodak XR-2 X-ray film and used to locate the positions of the radioactive bands corresponding to the L7 and L12 ribosomal proteins. The bands were cut from the gels and oxidized with 0.3 ml of 40% ^ 02 in the presence of 2% NH^OH for 18 hr at 37°C. Residual H ^ was neutralized by the addition of 0.75 ml of catalase (4 mg/ml solution of catalase in 10 mM Tris, pH 7.4) and the amount of radioactivity associated with the L7/L12 proteins determined directly by scintillation counting in 20 ml of 3a70B preblended liquid scintillation cocktail (Research Products International Corp.)- The relative differential rate of L7/L12 synthesis ^L7/L12^ w a s calculated as the ratio of pulse-labelled radioactivity to carrier cell labelled radioactivity in the L7 and L12 proteins com-pared to the same ratio in total protein as determined by TCA precipitation of aliquots of samples prior to acetic acid extraction. PART A: EXPRESSION OF RNA POLYMERASE AND RIBOSOME COMPONENT GENES IN MUTANTS OF ESCHERICHIA COLI HAVING CONDITIONALLY DEFECTIVE RNA POLYMERASES SUMMARY' The expression of the genes coding for the 3 and 3' subunits of RNA polymerase, ribosomal RNA, ribosomal proteins and B-galactosidase was investigated in strains carrying conditionally lethal mutations affecting either RNA polymerase core assembly or RNA polymerase enzyme activity. The mutant strain XH56 produces a temperature sensitive 3* subunit and at 42cfc is defective in RNA chain initiation; consequently, little or no transcript-ion occurs at the restrictive temperature. A partial restriction, produced by shifting the strain to 39°C, resulted in a rapid five-fold increase in the transcription of the rpoB and C_ genes and in the synthesis of 3 and 3' subunit proteins for which they code. The RNA polymerase assembly-defective strains A2R7 and Ts4 exhibited a 1.5-2 fold increase in the transcription of the rpoB and C^  genes and in the synthesis of 3 and 3' subunit proteins after prolonged restriction. These results demonstrate (i) that regulation of the synthesis of the 3 and 3' RNA polymerase subunits is under these conditions primarily transcriptional rather than translational and (ii) that a stimulation of r_poB and C^  gene expression results from a restriction on RNA synthesis caused by either RNA polymerase inactivation or inhibition of its assembly. During restriction of the mutant strains, the transcription of ribosome component genes exhibited patterns which were similar to trans-cription of the rpoB and C^  genes, supporting the evidence that genes coding for RNA polymerase are cotranscribed with ribosomal protein genes; trans-cription of the lacZ gene was observed to decrease concomitant with the stimulation of the rpoB and C^  genes. INTRODUCTION The Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6.) is responsible for virtually all cellular RNA synthesis. The RNA polymerase is a complex enzyme consisting of four different subunits in the stoichiometry of c^BB' ( 8, 14, 145). The rpoA gene, coding for the a-subunit, is situated in a ribosomal protein gene cluster near 72 min of the 12. coli chromosome ( 63 ; all map positions are designated according to reference 4 ).and is cotranscribed with several ribosomal protein genes ( 63 ). The rpoB and C^  genes, coding for the g and 3' subunits respectively, are in the same transcription unit (36,74,83 ) and are situated adjacent to a ribosomal protein gene cluster near 88 min (83,140). Recent experiments indicate that the rpoB and C^  genes form a common transcriptional unit with two of the adjacent ribosomal protein genes ( 142); the organization is promotor, rplJ, rplL, rpoB and rpoC (142). The rpoD gene, coding for the a-subunit, has recently been located near the dnaG locus at 66 min (50,53,101 ); no ribosomal protein genes have been located in this region of the chromosome. The core enzyme components a,3 and g' are synthesized in relatively constant molar ratios during steady-state growth and nutritional shift conditions whereas the a-subunit appears to be synthesized at essentially the same rate under different growth conditions (6,20,35,60,61,92 ). Since the core enzyme subunits are normally as stable as bulk E. coli protein (61 ), the rates of subunit synthesis must therefore control the intracellular levels of these proteins. The regulation of the synthesis of the RNA polymerase subunits and its relationship to ribosomal protein synthesis, however, is poorly characterized. The isolation of strains containing conditionally lethal mutations in genes coding for RNA polymerase subunits has facilitated investigations of the regulation of subunit synthesis (18,73,95 ). Using such strains, it was possible to uncouple g and g* subunit synthesis from a synthesis, suggesting that the synthesis of these subunits is regulated at least in part by different mechanisms (133). Studies with a mutant strain containing a thermosensitive RNA polymerase unable to catalyze nucleoside triphosphate incorporation into RNA under restrictive conditions indicated that the regulation of g and g' subunit synthesis is transcriptional and is somehow related to a decrease in the ability of the enzyme to initiate general transcription. Furthermore, this regulation is related to the transcription of ribosomal protein genes near 72 min and 88 min on the chromosome (24,26 ). In this investigation, the use of temperature-sensitive mutants defective in RNA polymerase assembly or activity has allowed the correlation of g and g' subunit protein synthesis with transcriptional activity at the rpoB and C^  genes. This supports the evidence that the regulation of g and g' subunit synthesis is primarily at the level of transcription. The results indicate that a general decrease in overall transcription rather than accum-ulation of assembly intermediates is probably responsible for the stimulation of g and g* synthesis. During restriction of the mutant strains the trans-criptional patterns of ribosome component genes were perturbed in a manner similar to transcription of the rpoB and C^  genes, consistent with the idea that genes coding for RNA polymerase subunits and ribosome components are cotranscribed (142). In contrast, transcription of the gene specifying g-galactosidase was severely depressed under conditions where transcription of the g and g ' subunit genes was stimulated. MATERIALS AND METHODS Bacterial strains and growth conditions The isogenic wild-type and mutant strains, obtained from J. Miller and J. Kirschbaum, were derived by PI transduction of the respective mutations to E. coli strain X239 (F , his, metB, thi, bfe, purD, argH2, rpsL, lac) (7,3). The wild-type strain X240 (F~, his, metB, thi, rpsL, lac) was obtained using a PI lysate from a wild-type donor and selecting the arg+ pur+ transductants. The mutant strains Ts4 and XH56, in addition, have the respective temperature-sensitive mutations rpoC4(ts) and rpoC56(ts) - . in the gene coding for the 3' subunit of RNA polymerase. The mutant A2R7 strain contains a double mutation, having both the .temperature-sensitive and spontaneous rifampicin resistance mutations rpoB7, rpoB2(ts) in the gene coding for the 3' subunit of RNA polymerase. Cultures were grown in M9 minimal salts medium supplemented with 0.2% glucose or 0.2% glycerol _3 (and 10 M isopropyl B-D-thiogalactoside, IPTG) as carbon and energy source and L-methionine (50 yg/ml), L-histidine (50 yg/ml), and thiamine (0.5 yg/ml) at the permissive temperature of 30 C (73). Cellular mass accumulation was determined by measuring the absorbance at 460 nm (A ). Cellular RNA 460 accumulation was monitored as the A„,n of the acid-insoluble, alkali-260 labile material .; Prior to experimentation, cultures were grown for at least 10 exponential cell doublings. At an A^Q of 0.25-0.35, the cultures were shifted to the non-permissive temperature of 42°C unless otherwise indicated. Protein labeling and quantitation of (3 and 3' subunits of RNA polymerase Cellular proteins were radioactively labeled at 30°C and at various o 14 times following a temperature shift to 42 C by the addition of [ C]leucine (sp. act. 348 mCi/mmole; 1.0 yCi/ml) to 5 ml sample volumes. Incorporation was terminated after 3 min by the addition of excess (50 yg/ml) non-radioactive L-leucine. Samples were further incubated for 2.5 min to allow for completion of nascent polypeptide chains. The cells were harvested and lysates were prepared for sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Cellular proteins were separated using 1.5 mm thick SDS slab gels, 7.5% in polyacrylamide, with 3% stacking gels. Gels were run at 25 ma for 7-8 hr, after which the 3 and 3' subunits had migrated 6.5-7.0 cm into the gels and were separated by about 2.5 mm. The gels were dried and autoradiograms were obtained using Kodak XR-2 X-ray film. Quantitation of radioactivity in the 3 and 3' region of the gels was obtained by scanning the autoradiograms with a Quick Scan Jr. scanner (Helena Institute, Beaumont, Tex.) equipped with an integrator. The areas of the 3 and 3' peaks were determined, corrected for different amounts of total radioactivity applied to the gels, and compared. In all instances, with the exception of strain XH56 at 42°C (see results), the amounts of total radioactivity applied to a gel varied by less than ±10%. Nucleic acid preparation and hybridization Pulse-labeled RNA was prepared by exposing 10-20 ml samples of the 3 cultures to [5- H]uracil (sp. act. 26 Ci/mmole; 10 yCi/ml) for 1 min. The cells were rapidly cooled to 0 C in the presence of 10 M Na azide, harvested, lysed in SDS, and cellular RNA was extracted by the phenol method (30). The RNA preparations were appropriately diluted to a concentration of about one A^ q^/iiiI (equivalent to 50 yg of RNA per ml) and used for hybridization as described (24). The DNAs used for hybridizations were from phage A, from the special-ized transducing phages Adtrk, Adspcl, \ilv5, and Aplac5, and the composite colEl plasmids pJC701, pJC703 and pJC720 (19,24,30,31; the composite plasmids previously have been referred to as pcc701, pcc703, and pcc720 respectively). The A and Adtrk DNAs were employed to measure non-specific hybridization. The Adtrk DNA contains the aroE and trkA genes located near 72 min on the _E. coli chromosome. The Adspcl DNA, in addition to aroE and trkA, contains a cluster of 15 ribosomal protein genes from this same region of the chromosome (rpsD,E,F,H,K,M,Q; rplE,F,N; rpmD) and the gene coding for the ot-subunit of RNA polymerase (rpoA). The difference in radioactivity hybridized with Adtrk and Adspcl DNAs therefore represents specifically that mRNA which is homologous to the DNA of the ribosomal protein gene cluster and the rpoA gene. The A ilv5 DNA contains a rRNA transcription unit and hybridizes almost exclusivelystable rRNA (19,25). The A plac5 DNA contains the lacZ gene and hybridizes mRNA transcribed from the lactose operon DNA. Hybridizations of radioactivity to A ilv5 DNAs were corrected for non-specific binding of radioactivity by sub-tracting the radioactivity associated with A DNA. The composite plasmid pJC720 carries the genes coding for the 3 and P 1 subunits of RNA polymerase (rpoB and C respectively), located near 88 min on the E. coli chromosome. The pJC703 DNA contains, in addition to rpoB and C^ , the adjacent ribosomal protein gene cluster (rplA, J,K,L). Radioactivity hybridizing to pJC720 DNA represents mRNA specifically homologous to the DNA of rpoB and C^  The difference in radioactivity binding to pJC 703 and pJC 720 DNAs therefore represents that mRNA which is homologous to the adjacent ribosomal protein gene cluster (24 ). All values were corrected for non-specific hybridization to A DNA. The pJC 701 DNA contains the rpoB gene. When used in competition experiments with pJC 720 DNA, the pJC 701 DNA competes for the RNA sequences which are homologous to rpoB but not to rpoC. Thus, the reduction in hybridization of RNA to pJC 720 DNA in the presence of excess pJC 701 DNA is rpoB mRNA; the residual hybridization to pJC 720 DNA is rpoC mRNA. Further details are described elsewhere (24 ). 3 In the temperature-sensitive strain XH56, when labeled with ( H)uracil at 39°C, the amount of radioactivity incorporated into RNA is small because of the restriction on RNA synthesis. Consequently, the specific activity of the rRNA is too low to employ the standard rRNA hybridization to excess Ailv5 DNA. To overcome this problem, a double label technique was employed 3 where the hybridization of the H pulse-labeled rRNA could be compared to 32 32 a standard P RNA preparation. The standard P RNA was prepared from a culture of homogeneously-labeled _E. coli strain NF314 as previously described (25,32). Under standard hybridization conditions of DNA excess, 32 40% of the P radioactivity hybridized to 20 yg of Ailv5 DNA. To assay 3 for mRNA in the H pulse-labeled RNA preparations, samples were mixed with 32 portions of the P RNA and hybridized. The assay contained radioactive RNA, one A DNA filter and two Ailv5 DNA filters (5 yg of DNA per filter). 3 32 Incubation was for 18 hr at 67 C. The isotope ratio ( H)/( P) in the input RNA and in the RNA hybridized to the Ailv5 DNA filters was determined 3 and the H hybridization was corrected according to the efficiency of hybridization of the P radioactivity. All measurements were in triplicate 3 and in all cases a minimum of 10 CPM of each isotope was hybridized to each Xilv5 DNA filter. RESULTS RNA Synthesis in the RNA Polymerase Mutant Strains. Strains X240, XH56, Ts4 and A2R7 are isogenic strains of Escherichia coli differing only in the genetic constitution of the rpoB and C^  genes. Strain X240 is wild-type whereas strains XH56 and Ts4 carry the thermo-sensitive mutations rpoC56(ts) and rpoC4(ts) respectively and strain A2R7 carries the thermosensitive and rifampicin-resistant mutations rpoB7 rpoB2 (ts). At the restrictive temperature of 42°C, strain XH56 is defective in RNA synthesis (18,26,51)whereas strains Ts4 and A2R7 are defective in different steps of core RNA polymerase assembly (59,131,132).The accumulation of cellular mass and RNA in these strains following a temperature-shift from 30°C to 42°C is illustrated in Fig. 3. Within 3-5 minutes of a shift to 42°C, cellular RNA and, somewhat later, cellular mass ceased to accumulate in the activity defective strain XH56. In contrast, in the RNA polymerase assembly-defective strains Ts4 and A2R7 the accumulation of cellular mass and RNA increased essentially arithmetically following .the temperature shift. This suggests that RNA polymerase assembled before the temperature shift remains active but that little or no additional core RNA polymerase is assembled after the temperature shift. Synthesis of 3 and 3' Subunits of RNA Polymerase at Restrictive Temperatures. The measurement of 3 and 3' synthesis was undertaken to determine the effects of a temperature shift on the synthesis of the 3 and 3' subunits of RNA polymerase. Samples from cultures of each strain were radioactively labeled at 30°C and at times following a shift to 39°C or 42°C. Lysates were prepared and cellular proteins were separated by SDS-polyacrylamide TIME AFTER TEMPERATURE SHIFT (MIN) TIME AFTER TEMPERATURE SHIFT (MIN) Figure 3: Accumulation of cellular mass (A) and RNA (B) after a temp-erature shift to 42°C. Strains X240 , XH56(A), Ts4 (g), and A2R7 (•) were grown at the permissive temperature of 30°C. At time 0 (i) theQCultures were shifted to the non-permissive temperature of 42 C. Cellular mass was monitored as the A . - ' •• Cellular RNA was determined as the A of the acid-insoluble alkali-labile fraction of the cell mass. The broken lines extrapolate the pre-shift rates into the post-shift region. gel electrophoresis. The amounts of 3 and 3' subunits relative to total radioactivity applied to the gels were determined (Table II]). The wild-type strain X240 exhibited a slight decrease in the relative synthesis rate of the 3 and 3* subunits after a shift to 42°C. In the assembly-defective mutant strain Ts4, the relative synthesis rate of the 3 and 3' subunits was elevated about two-fold after a prolonged period of incubation at the restrictive temperature. In the assembly-defective strain A2R7, a transient decrease was followed by a slight increase in the relative synthesis rates of the 3 and 3' proteins. These observations are consistent with previously reported measurements (73,133)and indicate that a restriction in core RNA polymerase assembly over an extended period of time can cause an increase in the relative synthesis rates of the 3 and 3' subunit proteins. When strain XH56, defective in RNA polymerase activity, was shifted to 42°C, the inhibition of total protein synthesis was virtually complete within 10 min and by 30 min no radioactivity was detectable on the auto-radiogram in the vicinity of the 3 and 3' subunit proteins. (Note: staining with Coomassie blue demonstrated that the 3 and 3' subunit proteins were present in the extracts but, since they were not radioactively labeled had been synthesized prior to the temperature shift.) Shifting strain XH56 to 39°C rather than 42°C only partially inhibits the activity of RNA polymerase (26). In such a shift it was observed that the relative synthesis rate of the 3 and 3' subunits increased almost five-fold within 20 min and then decreased slightly. Thus it was apparent from these results that partially inhibiting RNA synthesis by restricting RNA chain initiation profoundly affected the synthesis of the 3 and 3' RNA polymerase subunit proteins. Table III: Relative rates of 3 and 3' subunit synthesis after a shift to restrictive conditions. Time, min after Relative rate of 3 and 3' Temperature temperature shift subunit synthesis in: X240 Ts4 A2R7 XH56 30*42 C 0 1.00 1.00 1.00 1.00 30 0.82 0.93 0.39 0.00 120 0.71 2.08 1.31 0.00 30*39°C 0 5 10 15 20 30 45 1.00 1.75 2.75 3.79 4.75 3.26 2.51 Cultures of X240, Ts4, A2R7 and XH56 were grown at 30°C, the permissive temperature. At zero time, the cultures were shifted to the indicated temperature. At 0 min (30°C control) and at 30 and 120 min after the shift to the elevated temperature, 5 ml samples were pulse-labeled with (l^C)leucine for 3 min. Non-radioactive leucine was added in excess and incubation was continued for 2.5 min to allow for completion of nascent polypeptide chains. The cells were harvested and lysates were prepared. Cellular proteins were separated by electrophoresis on 7.5% SDS-polyacrylamide slab gels. The amount of radioactivity corresponding to 3 and 3' peaks was determined by integration and corrected for the different amounts of radioactivity applied to the gels (from 1x10 to 2x10^ CPM). The relative rates of synthesis are standard-ized so that the rate in the 30 control for each strain represents 1.0. The absolute amount of labeled 3 and 3' subunit protein in strain Ts4 was estimated to be about 2.7 fold greater than in the parental :.*; X240 strain at the permissive growth temperature of 30°C. The relative rates of subunit synthesis in the 30°C controls, compared to a value of 1.0 for the wild type strain X240, were 2.0, 0.9 and 1.1 respectively for Ts4, A2R7 and XH56. Transcription of jrpoB and C and Ribosomal Protein Genes in RNA Polymerase Assembly-Defective Mutants. The patterns of $ and 3' subunit synthesis obtained for strains Ts4 and A2R7 during restriction presumably result from alterations either in the transcription of the rpoB and C^  genes or in the translation of the rpoB and £ mRNA. If regulation of 3 and 3' subunit synthesis is primarily at the level of transcription, the patterns of 3 and 3' subunit synthesis would be expected to correlate with the amount of transcription occurring from the rpoB and C^  genes. To examine this, samples from cultures and strains X240, Ts4 and A2R7 were pulse-labeled with (5-3H)uracil at 30°C and at various times after a shift to 42°C. Radioactive RNA was prepared and used for hybridizations to an excess amount of pJC 720 DNA containing the rpoB and C^  genes Transcription from the rpoB and C^  genes in the wild-type strain X240 remained essentially constant or increased slightly following the temperature shift (Fig. 4A). In the mutant strain Ts4, transcription from the rpoB and (] genes increased steadily during the incubation period at 42°C reaching a level about two—fold above the 30 C control level after 120 min (Fig. 4.B). The amount of transcription from the rpoB and C^  genes at the permissive temperature of 30°C was elevated in the mutant strain Ts4 as compared to the wild-type strain X240; the relative amounts of 3 and 3' subunit proteins were also elevated in strain Ts4 compared to the wild-type strain at the permissive temperature. This suggests that even at 30°C assembly of core RNA polymerase in strain Ts4 may be partially defective. Transcription of the rpoB and C^  genes in the rpoB mutant strain A2R7 exhibited an altered pattern in the first 30 min following a shift to the Figure 4: Transcription of RNA polymerase and ribosomal protein genes in core RNA polymerase assembly defective mutants. Transcription of the rpoB and £ genes (A) and the ribosomal protein genes near 88 min (• ) and 72 min (•) on the E^. coli linkage map following a shift to the non-permissive temperature. Cultures of the wild-type strain X240 (A) and the mutant strains Ts4 (B) and A2R7 (C) were grown at the permissive temperature of 30°C. At zero time, the cultures were shifted to the non-permissive temperature of 42°C. Samples were pulse-labeled at time 0 (30°C control) and at indicated 3 times after the temperature shift by incubation with (5- H)uracil for 1 min. Radioactive-labeled cellular RNA was prepared and various amounts were hybridized to 2 blank filters, 2 A DNA filters, 2 Adtrk DNA filters, 4 Adspcl DNA filters and 2 pJC720 DNA filters; or 2 blank filters, 2 A DNA filters and 2 pJC703 DNA filters as described previously (24, 30, 31 )• Hybridizations to pJC720 and pJC703 DNA filters were corrected for background by subtracting the radioactivity binding to A DNA filters. The Adspcl hybridizations were corrected 4 similarly using Atrk DNA filters. The inputs ranged from 3.8 x 10 to 1.9 x 10^ CPM per 50 yl and each time point represents an experi-ment where 50, 100, 150 and 200 yl inputs were hybridized. The 30° control samples gave hybridizations to Adspcl DNA of 1.80, 2.10, and 1.76% of the input radioactivity respectively for X240, Ts4, and A2R7. The radioactivity hybridizing to the ribosomal protein gene cluster near 88 min was determined as the difference in hybridization to pJC703 and pJC720 DNAs. The results shown are normalized so that 100% represents the 30°C control hybridization to Adspcl DNA for each strain, as stated above. TIME AFTER TEMPERATURE SHIFT (MIN) non-permissive temperature (Fig. 4C). After 15 min, the level of rpoB and CI transcription was decreased slightly relative to the 30°C control level; at times after 30 min the amount of transcription gradually increased to a final level about 1.5-fold greater than the control. Although the apparent decrease at 15 min may be artifactual, the same pattern has been observed in independent experiments. In addition, previous investigators have also observed a decrease in the synthesis of 3 and g' subunit protein during this period (133). Because of the proximity of the RNA polymerase genes and ribosomal protein genes and because of the physiological relationships between transcription elements and translation elements, the transcription of ribosomal protein genes located at 72 min and 88 min was examined in the assembly-defective mutants during incubation at the non-permissive temperature. This was achieved by hybridizing the pulse-labeled RNAs to Adspcl and pJC 703 DNAs. In the wild-type strain X240 and the mutant strain A2R7, the ribosomal protein genes near 72 min and 88 min were essentially unaffected by the shift to 42°C. In strain Ts4, a slight increase in the level of ribosomal protein gene transcription was observed after 120 min at the restrictive temperature. RNA Polymerase Gene Expression in a Mutant Defective in Initiation of RNA Chains. A mutant defective in RNA polymerase activity exhibited properties substantially different from mutants defective in RNA polymerase assembly. When shifted to 42°C strain XH56, carrying the thermosensitive mutation rpoC56(ts), almost immediately ceased accumulation of RNA (Fig. 3). This cessation appears to result in vivo from an inhibition of RNA chain initiation rather than RNA chain elongation (26). After the initial few minutes at 42°C, essentially no RNA was synthesized and con-sequently, the effect of such a restriction on the general pattern of transcription of the chromosome cannot be determined directly. However, at temperatures several degrees below the absolutely restrictive temperature, initiation of RNA transcripts is only partially inhibited and RNA continues to accumulate, although at a somewhat reduced rate. Strain XH56 was grown at 30°C in glucose or glycerol minimal medium and at time zero shifted to 39°C. At various times, portions of the 3 14 cultures were labeled with ( H)uracil for 1 min or ( C)leucine for 3 min. In the respective samples RNA was prepared for hybridization or protein extracts were prepared for SDS gel electrophoresis. The imposition of a partial restriction in glucose minimal medium resulted in a four to five-fold increase in the relative synthesis rate of 3 and 3' proteins during the initial 30 min at 39°C (Table III). During the same time interval the relative level of transcription from the rpoB and C_ genes was elevated about five-fold (Fig. 5; 26). Using a competition hybridization assay it was possible to show that transcription of the rpoB and rpoC genes increased coordinately (Fig. 6 ); this result was expected since these two genes are in the same transcription unit (36,74,83,142 ). A similar restriction carried out in glycerol minimal medium resulted in a five to six-fold increase in the level of transcription of the rpoB and _C genes (Fig. 5 ). The transcription of genes specifying the RNA and protein components of the ribosome was also examined during the partial restriction of strain XH56 (Fig. 5 ). In both the glucose and glycerol-grown cells, the trans-Figure 5: Transcription of RNA polymerase ribosome component and lacZ genes in mRNA polymerase activity defective mutant. Transcription of rpoB and genes (• ), the ribosomal protein genes at 88 min (H) and 72 min (+), the lacZ gene (•) and the rRNA genes ( A ) following a shift of the activity mutant strain XH56 to a semi-permissive temperature. Cultures of strain XH56 were grown in glucose (A and B) or glycerol and IPTG (C and D) at the permissive temperature of 30°C. At time 0 min, the cultures were shifted to the semi-permissive temperature of 39 C. RNA was pulse-labeled with (5- H)-uracil and prepared for hybridizations at time 0 and at indicated times after the temperature shift. Hybridizations to determine trans-cription of the rpoB and jC genes and ribosomal protein genes (B and D) are described in Fig. 4 . Transcription from the lacZ gene was determined by hybridizing to Aplac DNA. The input RNAs contained from 1.7 x 105 to 2.4 x 104 CPM per 50 yil. The 30°C control prepara-tion gave hybridizations to Adspcl DNA of 2.07 and 1.78% respectively for the glucose and glycerol grown cells. The values are normalized so that the 30°C control Adspcl hybridizations represent 1.0. Trans-cription of the rRNA genes (A and C) was determined by using a double label technique as described. The 30°C control samples gave values of between 20 and 25% when hybridized to Ailv5 DNA. The results are standardized so that 1.0 represents the hybridization of the 30°C control preparations to Ailv5 DNA. 0 20 40 60 TIME AFTER TEMPERATURE SHIFT (MIN) \ Figure ft: Relative transcription of i;poB and rpoC genes In an RNA polymerase activity defective mutant. Relative transcription from rpoB and rpoC in the activity mutant XH56 after a temperature shift to 39°C in glucose minimal medium. Pulse-labeled RNA prepared at time 0 min (A) and at 30 min (B) after a temperature shift to 39°C (see Fig. 5 ) were used in competition hybridizations of pJC720 and pJC701 DNAs. Each hybridization 5 4 contained 100 yl of RNA (containing 3.33 x 10 and 6.18 x 10 CPM respectively for the 0 min and 30 min preparations), 2 A DNA filters and 2 pJC720 DNA filters. RNA sequences homologous to rpoB were competed from pJC720 DNA by including pJC701 DNA filters in increasing numbers in the hybridization assays. The values are expressed as the percent decrease of radioactivity binding to pJC720 DNA relative to hybridizations to pJC720 DNA in the absence of competitor DNA. Therefore, at excess pJC701 DNA concentrations, the reduction of RNA hybridization to pJC720 DNA represents rpoB mRNA and the residual hybridization is rpoC mRNA. 3 4 0 1 2 EQUIVALENTS OF COMPETITOR DNA cription of ribosome component genes was perturbed in a manner similar to the transcription of the RNA polymerase genes. Following the temperature shift to 39°C, the amount of transcription from rRNA and ribosomal protein genes increased to levels about 1.2-1.5 fold greater than the 30°C controls and then decreased, after 60 min, to approximately the pre-shift values. In the wild type strain X240, a temperature shift to 42°C had virtually no effect on the transcription of ribosomal protein genes (Fig. 4 A) or rRNA genes (results not shown). Finally, the transcription of the lacZ gene, specifying the inducible enzyme B-galactosidase, was examined in glycerol-grown cells in the presence of the inducer IPTG. It was apparent that transcription of the lactose operon was extremely sensitive to the restriction. At 5 min the level of transcription was only about 10% of the pre-restriction level and may have represented runoff of RNA polymerase which had initiated transcription of the lactose operon during the first few minutes of the restriction. After 20 min at 39°C, when the relative transcription of the rpoB and C^  genes had increased about six-fold, transcription from the lactose operon was essentially undetectable. DISCUSSION Regulation of g and g' subunit synthesis. The temperature-sensitive mutant strain XH56 is unable to initiate transcription at non-permissive temper-atures, but at intermediate temperatures, initiation of transcription is only partially restricted (26 ). When this strain was shifted to a partial-ly restrictive temperature, the relative rate of transcription of the rpoB and C^  genes exhibited a dramatic increase; this was accompanied by a conco— relative mitant increase in the^synthesis of the g and g' subunit proteins (Fig. 5 and Table III). Similar results were obtained when another rpoC mutant strain, also having temperature-sensitive RNA polymerase activity, was shifted to restrictive temperature ( 72). The results are also in agreement with those obtained when a wild-type strain of IS. coli was treated with the transcription initiation inhibitor rifampicin.. These observations indicate that an inhibition of transcription initiation selectively stimulates the relative rate of rpoB and C^  transcription; the resultant increase in rpoB and C! mRNA production leads to a corresponding increase in the relative rate of g and g' subunit protein synthesis. The. temperature-sensitive RNA polymerase assembly mutant strains Ts4 and A2R7 are defective in different steps in the assembly of core RNA polymerase (59,131,132 ). At the non-permissive temperature, only RNA poly-merase molecules which have been assembled prior to the temperature shift are active in transcription. As cellular mass accumulation and cell division occur, the intracellular concentration of this active and pre-assembled RNA polymerase progressively decreases. Eventually, a concentration incapable of sustaining further growth is reached since these strains cannot form colonies when incubated at the non-permissive temperature of 42 C. As seen in Fig. 3, cellular mass and RNA continued to accumulate when strains Ts4 and A2R7 were shifted to 42°C, although the accumulations became arithmetic rather than exponential. This indicates that the RNA polymerase assembled prior to the shift retains most, if not all, of its activity. In strain Ts4, the restriction of RNA polymerase assembly led to a gradual increase in the relative rates of rpoB and C^  transcription to double the preshift rates, with a concomitant increase in the synthesis of 3 and 3' subunit proteins (Fig. 4' and Table III;133). In strain A2R7, the restriction of RNA polymerase assembly resulted in a transient decrease in rpoB and >C transcription, but the relative rate of transcription of these genes eventually increased to 1.5 times the preshift rate; the synthesis of 3 and 3' subunit proteins responded in a qualitatively similar fashion. Thus, restricting the assembly of RNA polymerase also stimulates the rate of rpoB and C_ transcription and relative synthesis rate of 3 and 3' subunit proteins. In summary, these results indicate that the regulation of 3 and 3' subunit synthesis under these conditions is primarily transcriptional. Furthermore, it is evident that a rapid partial inhibition of transcription leads to a rapid relative increase in rpoB and C^  transcription, whereas a gradual inhibition of transcription leads to a gradual and less dramatic increase in rpoB and C_ transcription. This is most easily explained by proposing a mechanism which couples rpoB and C^  transcription, in an inverse manner to the total transcription capacity of the cell. Such a proposal is quite different from previous models which invoke subunits or assembly intermediates as autogenous regulatory effectors (see ref. 121 for a review). Expression of other genes during the restriction. The transcription of the rpoB and C^  genes has been shown to be coordinate with, but five-fold less frequent than, the transcription of the adjacent ribosomal protein gene cluster during steady-state growth (24). Recent genetic experiments indicate that the rpoB and (] genes form a single transcriptional unit together with two of the adjacent ribosomal protein genes; the organization is promotor, rplJ, rplL, rpoB and rpoC (142). The reduced frequency in transcription between the ribosomal protein and RNA polymerase genes is presumably the result of an attenuator which terminates about 80% of the transcripts during exponential phase growth. It is further apparent that, under certain conditions, the expression of the rpoB and C^  genes may be dissociated from that of the adjacent ribosomal protein genes. For example, the transcription of the rpoB and C^  genes and the synthesis of the 3 and 3' subunit proteins appears to be insensitive to stringent control during a partial amino acid deprivation whereas transcription of ribosomal protein genes and the synthesis of ribosomal proteins is substantially reduced (11,25,31,89). Under these conditions, termination at the attenuator seems to be relaxed. Similarly, in vitro experiments using a coupled transcription-translation system suggest that ppGpp reduces the production of ribosomal proteins but not the 3 and 3' subunit proteins (82). A second condition which dissociates the expression of the rpoB and (] genes from the ribosomal protein genes has been achieved using the temper-ature sensitive RNA polymerase mutant strain XH56. A partial restriction of this strain stimulated transcription of the rpoB and C^  genes about five-fold whereas transcription of the adjacent ribosomal protein genes was stimulated only about two-fold (above;26). Similarly, the expression of the rpoB and C^  genes was dissociated from that of the adjacent ribosomal protein genes in studies with the transcription initiation inhibitor rifampicin. When a wild-type strain of _E. coli was subjected to low concentrations of rifampicin, the transcription of the rpoB and _C genes was stimulated about three-fold while transcription of the adjacent ribosomal protein genes was stimulated by only about 50% (9 ). In all instances, however, the frequency of transcription of the ribosomal protein genes remained greater than the frequency of transcription of the rpoB and C^  genes. These observations are consistent with the proposal of an attenuator, with unique physiological properties, being located between the rplL gene and the rpoB and C^  genes. The transcription of the lactose operon was found to drop to essentially undetectable levels in conjunction with the stimulation of rpoB and C^  transcription in strain XH56 (Fig. 5). This response is probably not due to exclusion of the inducer, IPTG, or to the catabolite repression system and could reflect either a competition for active RNA polymerase based upon promotor strength or functional heterogeneity in the pool of RNA polymerase ( 9 ). In either case, it is clear that the selective increase in transcription of rpoB and (2 and ribosomal genes occurs even when transcription of the lac operon, which has a very strong promotor, is severely depressed. PART B: REGULATION OF RNA POLYMERASE SYNTHESIS: CONDITIONAL LETHAL AMBER MUTATIONS IN THE 3 SUBUNIT GENE SUMMARY Amber mutations in the rpoB gene specifying the 3 subunit of RNA polymerase coupled with conditional amber suppressors were used to restrict the synthesis of core RNA polymerase in strains of Escherichia coli. Such a restriction stimulated transcription of genetic units containing RNA polymerase subunit genes. Within the L10 transcription unit (genetic structure: promotor » rplJ (L10), rplL (L7/L12), attenuator, rpoB (3), rpoC (3*), terminator), the initiation of transcription at the promotor was enhanced and termination at the transcription attenuator was relaxed. Transcription of the genetic unit containing the rpoA gene (a) was also enhanced. In the strain containing a non polar amber mutation, the syn-thesis rate of the 3' subunit protein during the restriction correlated with the level of transcription of the 3 and 3' genes. In contrast syn-thesis of L7/L12 ribosomal protein remained essentially unaltered in spite of the elevated levels of L: J /L12 mRNA. INTRODUCTION Ribosomes and RNA polymerase are important components of the transcrip-tion-translation apparatus. In _E. coli the synthesis of the protein subunits of these two important cellular components is interrelated. The rpoB and rpoC genes specifying the g and g' subunits of RNA polymerase are cotranscribed with the rplJ and rplL genes specifying the ribosomal pro-teins L10 and L7/L12 (38,84,142). The two ribosomal protein genes occupy the proximal position in this transcription unit with the promotor located about 370 nucleotides upstream from the start of the L10 structural gene. The length of the intergenic space between the L10 and L7/L12 structural genes is 69 nucleotides, whereas the length of the space between the L7/L12 and g structural genes is 325 nucleotides (Fig. 7; 112). During exponential phase growth of wild-type strains of _E. coli the L10, L7/L12,g and g' proteins are produced in relative stoichiometries of about 1:4:0.2:0.2 (20, 92,128). Transcription of the proximal portion of this genetic unit encom-passing the L10 and L7/L12 genes is about 5-fold more frequent than trans-cription of the distal g and g' genes; this implies that about 80% of the transcripts of the ribosomal protein genes are terminated in the vicinity of the intergenic region between the rplL and rpoB genes.(24,29). It is likely that this transcription termination mechanism along with regulatory mechanisms acting at the promotor site within the leader region play important roles in regulating the synthesis of these proteins. The bacterial strains MX515 and MX550 contain respectively a non-polar and a polar amber mutation in the rpoB gene specifying the g subunit of RNA polymerase (107). When cultured at 25°C these strains produce both the g and g ' subunit proteins because of efficient suppressor tRNAs which £P22 r P ° B rplL rplJ rplA I -c ^ H T - ^ PROMOTER TERMINATOR KILO BASES o Figure 7: Organization of the RNA polymerase 3 transcription unit. The RNA polymerase genetic unit located near 88 min on the bacterial chromosome contains two ribosomal protein genes, rplJ (L10) and rplL (L7/L12), and two RNA polymerase genes, rpoB (B) and rpoC (3'). Transcription is initiated at a promotor located about 360 nucleo-tides in front of the rplJ gene (112). There is some evidence to suggest the existence of either a transcription attenuator or RNA processing site in the leader region about 250 nucleotides from the transcription start site (unpublished results cited in ref.112). In addition, several base substitution mutations which reduce expression of this transcription unit have been sequenced in this region ( 40 ) . In wild-type strains of IS. coli about 80% of the RNA transcripts reading through the ribosomal protein genes appear to be terminated in the vicinity of the rplL-rpoB intergenic space (24,29. insert the amino acid serine at the UAG termination codon within the RNA polymerase 3 mRNA. The activities of the suppressor tRNAs in the two strains are temperature sensitive (108). At 25°C suppression is about 90% in the non-polar strain and about 40% in the polar strain. At 43°C suppression is reduced to less than 10% in both strains. When incubated at temperatures intermediate between the permissive and restrictive con-dition these strains respond to produce near normal amounts of core RNA polymerase^. In the non-polar strain this results in a large overproduction of 3' subunit protein whereas in the polar strain both 3 and 3' subunit proteins are produced in near normal amounts^. In this study we have investigated the pattern of expression of genes within the L10 transcription unit as well as other transcription units specifying ribosomal protein and RNA polymerase subunits during permissive and restrictive conditions. The restrictions stimulated transcription initiation and relaxed attenuation within the L10 operon. In spite of the elevated transcription of the L10-L7/L12 genes the synthesis rate of L7/L12 protein remained essentially unaltered during restriction. t See Oeschger, M. (1976) Fed. Proc. Abstracts 35, 1638. MATERIALS AND METHODS Bacterial Strains and Growth Conditions. The isogenic strains used have the genetic background trp(am), leu(am), LacZ(am), galK(am), galE, tsx, strA and relA. In addition, strain MX515 was supD 43,74, sueA, rpoB (CI, non polar amber) and strain MX550 was supD 43,74, sueB, rpoB (38, polar amber). The supD 43,74, mutations are temperature sensitive and the sueA and sueB mutations enhance suppression (108). The strains, obtained from M. Oeschger, were cultured in MOPS minimal medium supplemented with glucose (2 mg/ml), a synthetic mixture of 16 natural amino acids (minus cys, met, tyr, phe) (102)and thiamine (1 yg/ml) for a minimum of 20 hrs at 25°C prior to experimentation. All measurements were done at cell densities (A^q nm, 1 cm path) of less than 0.80. Under these conditions the doubling times of the cultures at 25°C were consistently 150-180 min. Pulse Labeling of Proteins. Cellular proteins were pulse-labeled by exposing 5 ml samples of cultures growing at 25°C or for various times following a shift to 43°C 35 to ( S)methionine (spec. act. 608 Ci/mmol; 2 yCi/ml). Incorporation was terminated after 3 min by the addition of 40 yg/ml of non-radioactive methionine and the samples further incubated for 2.5 min. Cell lysates were prepared and a constant amount of each electrophoresed in sodium dodecyl sulfate 7.5% polyacrylamide gels to separate cellular proteins (26,76). Autoradiograms of the dried gels were prepared using Kodak XR-2 X-ray film. The amount of radioactivity associated with the 3 and 3' subunit proteins were determined by scanning the autoradiograms. The areas of the 3 and 3' peaks were determined and expressed relative to the total radioactivity incorporated during the pulse period and normalized to 1.0 in the 25°C control samples. Samples (5 ml) from a culture of MX550 growing at 25°C and shifted to 43° for 90 min were, in addition, labeled for 5 min with ("^S)methionine (2 yCi/ml) followed by a 2.5 min incubation in the presence of 40 yg/ml of non-radioactive methionine. The cells were harvested by centrifugation 3 and combined with (4,5- H) leucine labeled carrier cells. The carrier cells were prepared by incubating 50 ml of an exponential phase culture of MX550 at 25° for over a generation in the presence of (4,5-^H) leucine (spec. act. 342 mCi/mmol; 2 yCi/ml). In this instance, non-radioactive 3 leucine was omitted from the growth medium to increase ( H) leucine incorporation. The cells were harvested by centrifugation, and portions 35 were combined with the ( S)methionine pulse-labeled samples. The mixed 35 3 S, H-labeled cells were resuspended in 0.3 ml of 0.05 M Tris, 0.01 M EDTA, pH 8.1 and disrupted by freeze-thaw lysozyme treatment (23) followed 2+ by two ten sec sonication treatments. The Mg concentration was adjusted to 0.2 M and proteins were extracted with 2 volumes of glacial acetic acid at 0°C for 1 hr. The soluble fractions were dialyzed for 18 hr against 8 M urea, 0.05 M g-mercaptoethanol. The extracts were separated by electrophoresis on 1.5 mm thick, 4% polyacrylamide slab gels in the presence of 8 M urea (78) for 4-5 hrs at 40 mA. The gels were fixed in 1 N HC1, rinsed with 7% acetic acid and dried and autoradiograms were obtained. The radioactive spots corresponding to L7 and L12 proteins were 3 35 cut from the dried gels, oxidized with peroxide, and the H and S radioactivity determined by scintillation counting. Labeling of RNA and Hybridizations. Samples of 20 ml were removed from cultures growing at 25°C or at various times after a shift to 43°C and exposed to (5-^H)uracil (26 Ci/ mmol; 10 yCi/ml) for 1 min. Incorporation was terminated by rapidly cooling the cells to 0°C in the presence of 10 mM NaN^. Total cellular RNA was prepared, diluted to an A„,~ of one (equivalent to a concentration zoU of about 50 yg of RNA per ml) and hybridized to the respective DNAs (24). The DNAs used for hybridization were from phage A, the specialized transducing phages Adtrk and Adspcl, and the composite plasmids pJC720, pSPll and pNF1564. The A and Adtrk DNAs were used to measure non-specific hybridization. The Adspcl DNA contains DNA sequences from near 72 min in the chromosome specifying 15 ribosomal proteins and the a subunit of RNA polymerase and hybridizes ribosomal protein mRNA and a mRNA ("spc mRNA"). The pSPll DNA contains chromosomal DNA sequences specifying a portion of the RNA polymerase a subunit and hybridizes "a mRNA". The pJC720 DNA contains DNA sequences from near 88 min on the chromosome which specify the C terminal portion of the L7/L12 gene and the entire $ and 3' genes and hybridizes predominantly "33' mRNA" (24). The pNF1564 DNA contains chromosomal DNA sequences specifying the C terminal portion of the L10 and N terminal portion of the L7/L12 genes and hybridizes "L10, L7/L12 mRNA" (29). RESULTS Synthesis of B and 3' Subunit Proteins. When cultures of strains MX515 and MX550 were shifted from 25°C to 43°C, synthesis of complete 3 subunit peptide essentially ceased. In the MX550 strain carrying the polar mutation, synthesis of the 3' subunit peptide was also drastically reduced. During this restriction, DNA, RNA and protein synthesis continued but little or no further core RNA poly-merase was produced. As cell mass accumulated and cell division occurred, the intracellular concentration of core RNA polymerase progressively decreased to produce a nearly linear rate of growth which was maintained for as long as 3 hrs. The relative synthesis rates of the RNA polymerase 3 and 3' subunit protein were estimated at various times following a shift of the respective bacterial strains from 25°C to 43°C by labeling portions of each culture 35 for two min with ( S)methionine. The 3 and 3' subunit proteins were separated from the bulk of radioactive protein by electrophoresis in sodium dodecyl sulfate - 7.5% polyacrylamide gels (Fig. 8) (26,76). In strain MX515 carrying the non-polar amber mutation in the 3 gene, the synthesis rate of 3 subunit protein was about five-fold reduced at 43°C whereas the synthesis rate of the 3' subunit protein was progressively increased about four fold above the 25°C control level (Table IV). In strain MX550 carrying the polar amber mutation in the 3 gene, the synthesis rate of 3' subunit protein was reduced about 2-3 fold. Thus the shift to 43° in the two respective strains resulted in cessation of core RNA polymerase synthesis because of a failure to produce completed 3 subunit protein and produced the expected polarity on the expression of the Figure 8: Autoradiograms of pulse labeled protein separated on sodium dodecyl sulfate-polyacrylamide gels. Samples (5 ml) of cultures of MX515 and 1-1X550 growing at 25°C or at various times after a shift to 43°C were exposed to (3^S)methionine for 3 min followed by a further 2.5 min incubation in the presence of excess non-radioactive methionine. Extracts were prepared and a constant amount of each was electrophoresed in sodium dodecyl sulfate-7.5% polyacrylamide gels ( 26,76 ). Strain MX515: (A) 25°C; (B) 43°C, 90 min; (C) 43°C, 180 min. Strain MX550: (D) 25°C; (E) 43°C, 90 min; (F) 43°C, 180 min. The data is further quantitated in Table IV. The 3 min pulse and 2.5 min chase somewhat underestimates the synthesis rates of the 3 and 3' proteins. This is probably minimal since these pulse chase times are short compared to the cell doubling times and since the degradation half life of excess 3 and 3' subunits is generally of the order of 20 min or more (73 ). 80 A B C D E F — # Table IV: Quantitation of 3 and 3' subunit synthesis Relative Rate of Protein Synthesis"^ " Bacterial Strain 3 3* 3 and 3' MX515 25°C 0.55 0.45 1.00 43°C 90 min 0.11 0.69 0.80 43°C 180 min 0.14 1.69 1.83 MX550 25°C 0.39 0.61 1.00 43°C 90 min 0.05 0.21 0.26 43°C 180 min 0.01 0.32 0.33 In order to quantitate the amount of radioactivity associated with the RNA polymerase 3 and 3' subunit peptides, the autoradiograms shown in Fig. 8 were scanned and the areas of the peaks corresponding to the 3 and 3' proteins determined by integration. Measurements were corrected for differences in the total radioactivity applied 4 4 to the gels (from 2.09 x 10 CPM to 5.51 x 10 CPM). The relative rates of total 3 and 3' subunit peptide synthesis were normalized to values of 1.0 for the strains growing at the permissive temperature of 25°C. In the two strains, at 25°C, the amount of total 3 and 3' protein was essentially the same and represented about 1% of the total protein synthesis rate. distally located rpoC gene specifying 3' subunit peptide. Transcription Patterns During RNA Polymerase Limitation. Incubation of strains MX515 and 550 at 43°C prevents the further synthesis of core RNA polymerase molecules and because of continued growth the number of RNA polymerase available for transcription of the bacterial chromosome is gradually depleted. The patterns of transcription in the two strains were examined under these conditions by labeling portions of 3 each culture with ( H)uracil for one min. The radioactive RNAs were purified and hybridized to specific DNA probes which contained sequences specifying various ribosomal proteins or RNA polymerase subunits (Fig. 9 and Table V-) (24,29). In strain MX515 carrying the non-polar amber mutation in the 3 gene, transcription of the distal 3 and 3' mRNA (pJC720 hybridization) increased gradually about four fold during the restriction. This result correlates with the observation that the amber mutation is non-polar and that the synthesis rate of 3' subunit protein increased about four fold during the restriction. The genes specifying ribosomal proteins L10 and L7/L12 occupy the proximal position in this transcription unit (142) . Trans-cription of L10 and L7/L12 mRNA (pNF1564 hybridization) increased only about two fold during the restriction. During growth at 25°C transcription of the proximal ribosomal protein genes in this unit was about seven fold more frequent than transcription of the distal RNA polymerase genes (i.e. compare RET' values of 1.48 and 0.18 in Table V). This implies that in this strain about 85-90% of the transcripts reading through the ribosomal protein genes are terminated in the vicinity of the rplL - rpoB inter-Figure 9: Hybridization of pulse-labeled RNA to DNAs specifying ribosomal proteins and RNA polymerase subunit proteins. Samples (20 ml) removed from the respective cultures growing at 25°C or at various times after a shift to 43°C were exposed to (5-^H)-uracil for 1 min. Total cellular RNA was prepared immediately and hybridized to the DNA probes: pJC720 (A, right panels); pNF1564 (• , right panel^ ; Adspcl (# , left panels); and pSPll (• , left panels). The radioactivity per 50 yl of the respective RNA preparations are listed in Table V . Increasing volumes of the RNA preparations (50 yg/ml) were hybridized to excess amounts of the specific DNAs which had been denatured and immobilized on nitrocellulose filters at a concentration of 167 fmoles per filter. Each RNA preparation was used for two hybridization series: (1) two blank filters, two A DNA filters, 2 Adtrk DNA filters, four Adspcl filters and two pJC720 DNA filters, and (2) two blank filters, two A DNA filters, two pSPll DNA filters and two pNF1564 DNA filters. Hybridizations were for 18 hr at 67°C in a final volume of 2 ml of 0.3 M NaCl, 0.03 M Na citrate. Following hybridization, filters were treated with RNase ( 24). Non-specific association of radioactivity to specific DNA filters was corrected by subtracting radioactivity associated with A DNA filters ( or Adtrk DNA filters for the Adspcl hybridizations). Hybridizations of RNAs from MX515 at 25°C (A) and at 43°C, 180 min (B) and from MX550 at 25°C (C) and at 43°C, 180 min (D). The average values of the duplicate or quadruplicate filters are illustrated. The slopes of the respective hybridization curves reflect the fraction of the input radioactivity which hybridizes specifically to the probe DNA. (For further details of the hybridization procedure, see ref. 24 ). The data is further quantitated in Table V . RADIOACTIVITY IN SPECIFIC HYBRIDS, (CPM/FILTER) Table y; Transcription of RNA polymerase and ribosomal protein genes. The data presented are summarized from the experiments presented in part in Fig. 9 . Input radioactivity and radioactivity in specific RNA-DNA hybrids are normalized to an input of 50 yl of radioactive RNA and percentages hybridized were calculated. The relative frequency of transcription (RFT) of the chromosomal DNA sequences represented by the respective hybridization probes was calculated using the relationship: (% of total transcription hybridizing to the specific sequences of the probe DNA) x (total molecular weight of protein specified by the specific sequences in Adspcl DNA) RFT = : : (% of total transcription at 25°C hybridizing to the specific sequence of Adspcl DNA) x (total molecular weight of protein specified by the specific sequences in the probe DNA) These RFT- values represent the frequency of transcription of a particular sequence relative to the frequency of transcription at 25°C of the sequences represented on the Adspcl DNA. It was assumed that Adspcl DNA specifies 260,000 and 40,000 daltons of ribosomal protein and a subunit protein respectively. The plasmid pJC720 specifies i 7,000 and 320,000 daltons of L7/L12 and 3 and 3' protein respectively. The plasmid pSPll contains the 1.4% Hind III fragment from Afus2 inserted into the Hind III site of pBR322 and specifies about 25,000 daltons of a subunit protein. The plasmid pNF1564 contains a 658 nucleotide EcoRI-Pst fragment from Arifd 18 inserted between the Eco RI and Pst sites of pBR322. This fragment begins in codon 23 of the L10 gene and extends to codon 57 of the L7/L12 gene and specifies about 23,000 daltons of L10 and L7/L12 protein. Table V: Hybridization of Pulse Labeled RNA to Specific DNA Probes. Hybridization Probe Adspcl ("spc" mRNA) pSPll ("a" mRNA) Bacterial Strain Input CPM in % of Radioact. Hybrids input R.E.T. CPM in % of Hybrids input R.F.T. MX515 (non polar) 25°C 4.25 X 1 0 t 748 1.76 1.0 74 0.17 1.16 43°C 90 min 2.87 X 10t 616 2.15 1.22 66 0.23 1.57 43°C 180 min 1.77 X 104 420 2.37 1.35 40 0.23 1.57 MX550 (polar) 25°C 4.67 X 1 0 4 832 1. 78 1.0 72 0.15 1.01 43°C 90 min 2.20 X 1 0 4 556 2.52 1.42 58 0.26 1.75 43°C 180 min 2.72 X 10 652 2.40 1.34 72 0.26 1.75 pJC720 ( "Rftt » mRNA) pNF1564 (L10-L7/L12 mRNA) MX515 (non polar) 25°C 43°C 43°C 25°C 43°C 43°C 4.25 X io4 148 0.35 0.18 86 0.20 1.48 90 min 2.87 X 1 0 4 276 0.96 0.50 104 0.36 2.67 180 min 1.77 X 10 238 1.34 0.70 74 0.42 3.11 1 (polar) 4.67 X 1 0 4 316 0.68 0.35 130 0.28 2.05 90 min 2.20 X 10t 266 1.21 0.62 166 0.76 5.57 180 min 2.72 X 104 296 1.09 0.56 190 0.70 5.13 .genie space (as compared to values of about 80% in other wild type strains of IS. coli) (24,29). This situation appears to be a normal component of the regulation of distal genes in this transcription unit. The restriction on RNA polymerase synthesis imposed on strain MX515 thus resulted in about a two fold relaxation in the normal mechanism terminating transcripts beyond the ribosomal protein genes as well as a two fold increase in the frequency of transcripts entering the proximal ribosomal protein genes. Prior to and following the restriction, pulse labeled 3 mRNA sequences represented respectively about 50% and about 60% of the total 33' mRNA (values determined by competition hybridization as described in ref. 24). Since the 3 and 3' genes are approximately of equal length, these measure-ments support the suggestion that the termination occurs at an attenuator site located early or in front of the 3 subunit gene. Following the restriction, termination at this site is gradually reduced. The slight increase in the fraction of 3 mRNA further suggests that the amber mutation in strain MX515 may exhibit a very low level of polarity. In addition to 3 and 3' subunits, core RNA polymerase also contains two copies of the a subunit. The restriction of strain MX515 prevented further assembly of core polymerase by blocking the synthesis of 3 subunit protein. The influence of this restriction on the transcription of the rpoA (a subunit gene) and a cluster of ribosomal protein genes located near 72 min on the genetic map was also examined by hybridization to pSPll and Adspcl respectively. The restriction resulted in only a modest 30-40% increase in the transcription frequency of these genes (Table 2). The transcription results obtained with strain MX550 carrying the polar amber mutation in the 3 subunit gene were qualitatively similar to the results obtained with strain MX515. At 25°C the efficiency of sup-pression of the polar amber mutation was only about 40%; this resulted in somewhat elevated levels of transcription of the proximal L10, L7/L12 mRNA (pNF1564 hybridization) as well as the distal 33' mRNA (pJC720 hybridization) and was presumably necessary to ensure adequate production of core RNA polymerase components. When suppression was further reduced by incubation at 43°C, L10 - L7/L12 mRNA transcription increased nearly three fold (clearly demonstrating an increased transcriptional expression of this genetic unit), whereas 33' mRNA transcription increased only 60-80%. The relative abundance of radioactive RNA sequences transcribed respectively from (i) the 3 gene and (ii) the 3' gene was again determined using a competition hybridization assay (see reference 24 for assay). At 25°C about 55-60% of the total 33' mRNA represented 3 mRNA sequences. After 90 min at 43°C this fraction was increased to about 80% (data not illustrated). This result indicated that the relatively small 60-80% increase in total 33' mRNA in the polar MX550 strain following a shift to 43°C was a consequence of either reduced transcription of the 3' gene caused by termination of most RNA transcripts in the vicinity of the amber mutation within the 3 subunit gene or a greatly reduced half life of mRNA beyond the site of the UAG termination codon. The transcription of the RNA polymerase a subunit gene and the ribosomal protein gene cluster located near 72 min on the bacterial chromosome was near normal at 25°C and stimulated about 70% and 40% respectively by the restriction. Synthesis of L7/L12 Ribosomal Protein. The observed two to three fold increase in the transcription of the proximal rplJ and rplL genes during restriction of suppression in the amber mutant strains led us to examine the relative synthesis rate of L7/L12 ribosomal protein under these conditions. The ribosomal proteins L7 and L12 are both specified by the rplL gene. The two proteins have identical amino acid sequences and differ only by the presence of an acetyl substitution of the amino group of the N terminal serine of L7 (138). A culture of the MX550 polar amber strain was labeled for 5 min with (^S)methionine at 25°C and after 90 min at 43°C. Protein extracts were prepared and separated in urea polyacrylamide gels (Fig.io) (78). The L7 and L12 proteins were cut from the dried gels and the associated radioactivity was determined. It was apparent that although the re-striction at 43°C stimulated transcription of the L7/L12 gene by two to three fold, there was no increase observed in the synthesis rate of L7/ L12 protein. This implies the existence of a mechanism which restricts translation of L7/L12 mRNA. It was apparent from the autoradiograms and confirmed from the direct radioactivity determination that in strain 35 3 MX550 about 70-80% of the S and H-radioactivity was present in the acetylated L7 form of the protein. Figure 10: Autoradiograms of pulse labeled proteins separated on urea polyacrylamide gels. Samples from a culture of strain MX550 growing at 25°C and shifted to 43°C for 90 min were labeled with ("^S)methionine for 5 min and further incubated for 2.5 min in the presence of excess non-radioactive methionine. The cells were harvested, mixed with 3 ( H)leucine labeled carrier cells, and extracts were prepared. A constant amount of each extract was electrophoresed in urea-4% polyacrylamide gels. (A) Standard preparation of IS. coli B/r 35 labeled with ( S)methionine to illustrate the positions of L7 and L12 proteins; (B) MX550. labeled at 25°C; (C) MX550 labeled after 90 min at 43°C. The data is further quantitated in Table VI . Again it is assumed that this labeling procedure estimates the \ synthesis rate of L7/L12 protein. A rapid and extensive degrad-ation of newly synthesized L7/L12 protein would result in an underestimate in the synthesis rate. B L 1 2 Table VI: Quantitation of the synthesis rate of L7 and L12 protein. ( 3 5S/ 3H) Isotope Ratios L7+L12 C Normalized Relative Ratio m^-ir. 4. • a I 7 i n o b Total Protein Total Protein L7+L12 MX550 25°C 4.22 5.11 1.21 1.00 MX550 43°C 90 min 3.89 4.14 1.06 0.88 Isotope ratio determined following trichloroacetic acid precipitation of a portion of the samples prior to lysis. k Isotope ratio determined following peroxide oxidation of chips cut from dried gels (lanes B and C from Fig.10 )• For simplicity these ratios represent the summed data for the two proteins. In the 25°C control, the L7 and L12 ratios were essentially identical (5.13 and 5.06) with about 80% of the protein in the L7 form. For the sample obtained from the 43°C culture about 70% of 35 the pulse labeled S-radioactivity was associated with L7 and about 30% with L12. The quotient of the isotope ratios is a measure of the synthesis rate of L7+L12 protein relative to the total protein synthesis rate. These measure-ments are normalized to a value of 1.0 for the 25°C control sample. DISCUSSION From the results presented here and elsewhere it is apparent that a limitation in the number of RNA polymerase molecules capable of trans-cribing the bacterial chromosome results in the enhanced expression of the rpoB and C genes (9,26,54,72,73,85,107,121). The restriction in the number of active polymerase molecules can be imposed rapidly by either adding the antibiotics which inhibit RNA polymerase activity or using strains which are temperature sensitive for RNA polymerase activity, or imposed more slowly by inhibiting synthesis of new core RNA polymerase in mutants defective in either subunit assembly or in the synthesis of one or more of the subunit proteins. Such restrictions result in enhanced transcription of the rpoB and C^  genes and a parallel increase in the synthesis of the 3 and 3' subunit proteins in all cases except where translation is blocked by the presence of amber mutations. The increased transcription of the rpoB and C^  genes in the experiments reported here resulted from (i) more RNA transcripts entering the structural genes of the genetic unit from the promotor-leader region and (ii) a relaxation of the mechanism normally responsible for high frequency termination of RNA transcripts in the vicinity of the rplL^rpoB intergenic space. In strain MX550 carrying the polar amber mutation the effect of relaxed attenuation was obscured by polarity whereas in strain MX515 carrying the non-polar amber mutation transcription read through increased from 12% to at least 23% during the restriction. Measurements of the relative rate of synthesis of ribosomal protein L7/L12 during the restriction indicated that it did not increase appreciably in spite of the fact that transcription of the rplJ and Ij genes was enhanced two to three fold. This is consistent with the previous observations that rifampicin mediated restrictions failed to stimulate the synthesis of L7/L12 protein (54). In addition it has been further demonstrated that plasmid mediated amplification of the 3 trans-cription unit results in a six fold elevation in rplJ and L^  mRNA trans-cription but only a 30% increase in the synthesis rate of the L7/L12 protein (29). Together these results suggest the existence of a mechanism which limits translation of excess L7/L12 mRNA in order to ensure a balance between the production of L7/L12 protein and the overall rate of ribosome biosynthesis and at the same time to allow for some dis-sociation between L7/L12 synthesis and 3 and 3' synthesis. The transcriptional response of the rpoA gene, located near 72 min and specifying the a subunit, to the cessation of 3 subunit synthesis was clearly less dramatic (Fig. 9. and Table V) than the response observed within the RNA polymerase 3 transcription unit. This may be related to the fact that a protein is normally present in about a two-fold excess over the amount required for core RNA polymerase (35):. The transcription of the ribosomal protein gene cluster located near 72 min (which contains the ct subunit gene) was stimulated about 30—40%. Most or all of this stimulation probably results from increased transcription of the genetic unit containing the a subunit gene (rpoA) and several ribosomal protein genes (rpsK, rpsD, rplQ and probably rpsM) (63). In summary, we suggest that the synthesis rate of the 3 and 3' subunits of RNA polymerase is related to the overall transcriptional capacity of the cell. When this capacity to transcribe is reduced, transcription of the genetic unit containing the rpoB and C^  genes is preferentially enhanced; 33' mRNA is translated with normal efficiency whereas translation of the excess L7/L12 mRNA is restricted in order to maintain balanced production of ribosome components. PART C: TRANSCRIPTIONAL AND POST TRANSCRIPTIONAL CONTROL OF RIBOSOMAL PROTEIN AND RNA POLYMERASE GENES SUMMARY A partial restriction of RNA polymerase activity has been used to dissociate the coordinate synthesis of ribosomal proteins and subunits of RNA polymerase and to identify transcriptional and post-transcriptional control signals which regulate the expression of these component genes. Within the L10 operon (which has the genetic organization promotor > rplJ (L10), rplL (L7/L12), attenuator, rpoB (3), rpoC (3'), terminator) the restriction caused an elevation in the relative frequency of trans-cription initiation at the promotor and, in addition, relaxed trans-cription termination at the attenuator. Together these selectively increased transcription of the RNA polymerase 3 and 3' genes. Trans-cription within the operon containing four ribosomal protein genes and the RNA polymerase a gene was also enhanced by unknown mechanisms whereas transcription within operons containing only ribosomal protein genes was virtually unaffected by the restriction. It was thus concluded that the mechanisms controlling transcription initiation and attenuation in operons containing RNA polymerase subunit genes are coupled to the global rate of RNA synthesis. By introducing the composite Col El plasmid pJC701 carrying the proximal portion of the L10 operon including the 3 subunit gene, it was possible to achieve a 10 fold and a 30 fold range in the transcription intensity of the genes specifying L10-L7/L12 and 3 respectively. Under these conditions the relative synthesis rate of L7/L12 and 3 protein varied by less than two fold and by about 15 fold respectively. These observations demonstrate the existence of a post-transcriptional mechanism which severely restricts translation of excess L7/L12 and probably also L10 ribosomal protein mRNA; this mechanism is probably important in maintaining the balanced synthesis of ribosome components under conditions where their mRNA levels are dissociated. Furthermore, the observed reduction in the translation efficiency of 3 subunit mRNA may be related to an inhibitory effect caused by accumulation of RNA polymerase assembly intermediates. INTRODUCTION The genetic organization and the regulation of the genes specifying the protein components of the ribosome and of RNA polymerase is complex and interrelated (for a review see Reference 105 ). In Escherichia coli the RNA polymerase a subunit gene and the 3 and 6' subunit genes are in two separate transcription units; each of these units also includes ribo-somal protein genes (15,38,84,103,142 )• The genetic organization of the L10 transcription unit located near 88 min on the chromosome is promotor (PL1Q), rplJ (L10), rplL (L7/L12), attenuator, rpoB (3), rpoC (3'), terminator (5,24,112 ). During balanced growth the transcription attenuator terminates about 80% of the RNA transcripts and is apparently responsible at least in part for the observed 5:1 stoichiometry between ribosomes and RNA polymerase (24). The rpoA (a subunit) gene is located within the major cluster of ribosomal protein genes near 72 min and is cotranscribed with rpsM (S13), K (Sll), D (S4) and rplQ (L17) (63,103. Although the synthesis of ribosomes and RNA polymerase is normally coordinate, under certain conditions of stress this coordination can be dissociated. For example the stringent control system modulates the trans-criptional intensity of ribosome component genes during periods of amino acid insufficiency; at the same time the transcriptional intensity of the 3 and 3' RNA polymerase subunit genes remains virtually unaffected (89 ). This apparent paradox between genetic and physiological observations can be explained by a reduction in the transcription initiation frequency at the L10 promotor and a concomitant relaxation of attenuation. A second means of dissociation is through a reduction in RNA polymerase activity (26,54). This selectively stimulates the transcriptional in-tensity of the 3 and 3' RNA polymerase subunit genes. In this communi-cation some of the regulatory mechanisms influencing transcription of the various ribosomal protein and RNA polymerase genes and the subsequent translation of these mRNAs, with special emphasis on genes within the L10 transcription unit, are examined during a restriction of RNA poly-merase activity. The results demonstrate (i) that the activity of RNA polymerase influences the transcription initiation and attenuation in the operons containing the 3 and 3' and the a RNA polymerase subunit genes and (ii) that post transcriptional mechanisms restrict the translation of excess L7/L12 mRNA and to a lesser extent 3 mRNA produced under these conditions. MATERIALS AND METHODS Bacterial Strains and Growth Conditions. The bacterial strain XH56, an F , his, thi, metB, strA, lac, rpoC56(ts) derivative of _E. coli X239, obtained from J. Miller, contains a temperature sensitive mutation in rpoC which renders RNA polymerase inactive at 42°C ( 18,26). Strain XH56-701 was obtained by transformation of XH56 with the plasmid pJC701; the bacterial substitution on this plasmid contains the proximal portion of the L10 transcription unit including the promotor, rplJ, rplL and rpoB (dominant rifampicin resistant allele) but not the distally located rpoC gene ( 19,29). Cultures were grown in M9 minimal medium supplemented with glucose (2-6 mg/ml), methionine (50 yg/ml), histidine (50 yg/ml) and thiamine (0.5 yg/ml) at the permissive temperature of 30°C. Accumulation of cellular mass was monitored as the absorbance at 460 nm (A,^ ,.). Under these conditions, the generation times respectively 460 for XH56 and XH56-701 were 100 min and 150 min. Cultures were grown exponentially for at least 8 generations prior to experimentation. At an A^q 0.35-0.40 the cultures were shifted to 38.5°C to partially inactivate the RNA polymerase and therefore reduce transcription. RESULTS Genetics and Regulation. The bacterial strain XH56 carries the mutation rpoC56 and produces a thermosensitive 6' subunit of RNA polymerase (18 )- At the restrictive growth temperature of 42°C RNA synthesis rapidly ceases due to an in-ability of the mutant RNA polymerase to initiate transcripts. At a some-what lower temperature of 38.5°, initiation is only partially inhibited and global RNA synthesis is reduced by about 50%. This partial restriction has the effect of specifically stimulating the transcription of the 3 and 3' RNA polymerase genes by 4-6 fold (26,85 )• To further amplify the consequences of regulatory events taking place during a partial restriction, strain XH56 was transformed with the composite ColEl plasmid pJC701. Transformants containing the plasmid remained temperature sensitive for RNA synthesis, indicating that the plasmid does not complement the chromo-somal rpoC56 mutation, and were resistant to the antibiotic rifampicin because of the dominant rifampicin resistant rpoB allele on the plasmid. In XH56-701 the portion of the L10 operon present on the plasmid, including the important regulatory sites, was amplified about 10 fold because of the copy number of the plasmid ( 29 ). Transcription with Operons Containing RNA Polymerase Genes. The expression of the complete chromosomal and the partial plasmid L10 transcription units was subjected to manipulation by temperature inactivation of the 3' RNA polymerase subunit. Samples from cultures of XH56 and XH56-701 were removed prior to and at various times after a temperature shift from 30°C to 38.5°C and incubated for one min with (3H) uracil to label nascent RNA transcripts with radioactivity. The RNAs were purified and hybridized to specific DNA probes to assay for mRNA transcribed from (i) the proximal L10 and L7/L12 ribosomal protein genes of the L10 transcription unit, (ii) the distal 3 and B' RNA polymerase genes, (iii) a cluster containing 15 ribosomal protein genes and the RNA polymerase a gene, located near 72 min on the bacterial chromosome and (iv) a portion of the RNA polymerase a gene alone. The relative frequencies of transcription of the respective sequences represented on the probe DNAs are presented in Table VII and Fig.11. In the non plasmid strain XH56 at 30°C, the L10 and L7/L12 genes and the separate cluster of ribosomal protein genes in the spc region of the chromosome were transcribed with nearly equal frequency. In contrast, the 3 and 3' RNA polymerase genes which are located distal to the L10 and L7/L12 genes were transcribed at about one-fifth the frequency. This implies the existence of a transcription attenuator in the vicinity of the L7/L12-B intergenic space which terminates about 80% of the RNA transcripts. The RNA polymerase a gene was transcribed nearly twice as frequently as the spc cluster as a whole. In other strains it was trans-cribed at essentially the same frequency; the reason for this difference is not known but may be related to the rpoC56 mutation. Following the partial inactivation of RNA polymerase activity, transcription of the L10 operon was enhanced. The relative frequency of transcription of the proximal ribosomal protein genes and the distal RNA polymerase genes was increased about two-fold and four-fold respectively. Thus, the elevated intensity of 3 and 3' gene transcription results from (i) an increase in initiation of transcription of the operon and (ii) a two-Table VII: Relative frequences of transcription of ribosomal protein and RNA polymerase genes. Samples (15 ml) of cultures growing at 30°C or for various times after a temperature shift to 38.5°C were exposed to (5-%)uracil (specific activity 25 Ci/mmol; 10 yCi/ml) for one min. Radioactive RNA was prepared and hybridized to constant and excess amounts of specific and nonspecific DNAs as described (24 ). Briefly, the DNAs used for hybridizations were from phage A, from the specialized transducing phages Adtrk and Adspcl, and from the composite plasmids pJC720, pNF1564 and pSPll. The A and Adtrk DNAs were employed to measure nonspecific hybridization. The A dtrk DNA contains the aroE and trkA genes located near 72 min on the JS. coli genetic map. The Adspcl DNA, in addition to aroE and trkA, contains a cluster of 15 ribosomal protein genes and the rpoA gene from the same region of the chromosome which specify about 260,000 daltons of ribosomal protein and the entire 40,000 daltons of the RNA polymerase a subunit. The pSPll DNA contains a Hind III fragment from the rpoA gene which specifies about 25,000 daltons of the RNA polymerase a subunit. The pJC720 DNA contains the C terminal portion of rplL and the entire rpoB and C^  genes, located at 89 min on the 12. coli chromosome, which specify about 7000 daltons of L7/L12 and 320,000 daltons of the RNA polymerase 0 and 3' subunits respectively (29). The pNF1564 DNA contains a 658 nucleotide EcoRI-Pst fragment which extends from the C terminal portion of the rplJ gene to the N terminal portion of the rplL gene. This fragment specifies about 23,000 daltons of L10 and L7/L12 protein (29). The relative frequency of transcription (RFT) of the chromosomal DNA sequences represented by the respective hybridization probes was calculated using the relationship: (% of total transcription hybridizing to the specific sequences of the probe DNA) x (total molecular weight of protein specified by the specific sequences in Adspcl DNA) RFT = ' (% of total transcription in strain XH56 at 30 C hybridizing to the specific sequence of Adspcl DNA) x (total molecular weight of protein specified by the specific sequences in the probe DNA). These RFT values represent the frequency of transcription of a parti-cular sequence relative to the frequency of transcription at 30°C in strain XH56 of the sequences represented on the Adspcl DNA. The values in parenthesis represent the relative frequency of transcription of the 3 subunit gene alone in strain XH56-701. It has been demonstrated using competition hybridization (24) that both prior to and following the shift about 85% of the total radioactivity hybridizing to the pJC720 DNA probe represents 3 mRNA (derived from both the plasmid and chromosomal genes) and 15% represents 3' mRNA (derived only from the chromosomal gene). Table VII: Relative frequences of transcription of ribosomal protein and RNA polymerase genes. Input CPM in % of RFT CPM in % of RFT radioact. Hybrids Input Hybrids Input L10-L7/L12 mRNA 63' mRNA XH56 30° 1.30 X 1 0 221 0.17 0.92 598 0.46 0.18 38.5°-5' 8.40 X 225 0.28 1.52 699 0.87 0.33 38.5°-10' 6.24 X 193 0.31 1.68 817 1.31 0.50 38.5°-20' 5.28 X 10t 132 0.25 1.36 913 1.73 0.66 38.5°-30' 4.08 X 10t 127 0.31 1.68 702 1.72 0.66 38.5°-45' 4.56 X 10t 146 0.32 1.74 666 1.46 0.56 38.5°-60' 4.96 X io4 119 0.24 1.30 536 1.08 0.41 XH56-701 30° 5.44 X 1 0 4 544 1.00 5.44 944 1.74 0.67(1. 14) 38.5°-5' 2.40 X 111 1.13 6.15 872 3.63 1.39(2. 35) 38.5°-10' 1.14 X 1 0 4 160 1.40 7.60 512 4.49 1.72(2. 93) 38.5°-20' 1.36 X 10t 208 1.53 8.31 800 5.88 2.25(3. 82) 38.5°-30' 1.74 X 1 0 4 304 1.75 9.51 1128 6.48 2.48(4. 22) 38.5°-45' 1.40 X 256 1.83 9.95 1192 8.51 3.25(5. 25) 38.5°-60' 1.23 X io4 280 2.28 12.40 1120 9.11 3.48(5. 92) Aspcl mRNA a mRNA XH56 30° 1.30 X 1 0 4 3120 2.40 1.00 507 0.39 1.95 38.5°-5' 8.40 X 10t 2010 2.50 1.04 402 0.50 2.50 38.5°-10' 6.24 X 1566 2.51 1.05 306 0.49 2.45 38.5°-20' 5.28 X 1 0 4 1336 2.53 1.05 275 0.52 2.60 38.5°-30' 4.08 X 1 0 4 1057 2.59 1.08 241 0.59 2.95 38.5°-45' 4.56 X 1 0 4 1140 2.50 1.04 319 0.70 3.50 38.5°-60' 4.96 X io4 1329 2.68 1.12 327 0.66 3.30 XH56-701 30° 5.44 X 1 0 t 1360 2.50 1,04 224 0.41 2.05 38.5°-5' 2.40 X 1 0 4 736 3.07 1.28 136 0.57 2.85 38.5°-10' 1.14 X 1 0 4 256 2.25 0.94 96 0.84 4.20 38.5°-20' 1.36 X K 336 2.47 1.03 112 0.82 4.10 38.5°-30' 1.74 X 10t 448 2.57 1.07 144 0.83 4.15 38.5°-45' 1.40 X 320 2.29 0.96 104 0.74 3.70 38.5°-60' 1.23 X io4 272 2.21 0.92 104 0.85 4.25 Figure 11: Transcription of genes coding for RNA polymerase subunits and ribosomal proteins. The relative frequency of transcription of DNA sequences specifying L10-L7/L12 mRNA (•), 3"B' mRNA ('•"), 6 mRNA alone (•) and a mRNA ( A ) are from data presented in Table VII and are norm-alized to a value of 1.0 for the frequency of transcription of the ribosomal protein gene cluster represented on the transducing phage Adspcl. The open symbols and stippled lines illustrate the relative frequency of transcription of the respective DNA sequences prior to the temperature shift from 30 38.5°C. Strain XH56 (left panel) and strain XE56-701 (right panel): for further details see Table VII. RELATIVE FREQUENCY OF TRANSCRIPTION to cj -h. o — o O Cn O O o O RELATIVE INCREASE IN TRANSCRIPTION RELATIVE FREQUENCY OF TRANSCRIPTION RELATIVE INCREASE IN TRANSCRIPTION fold relaxation in transcription termination at the attenuator (Fig.12). The partial restriction resulted in only a slight increase in transcription of the spc ribosomal protein gene cluster. However, transcription of the RNA polymerase a gene which is part of the spc gene cluster was stimulated about two-fold. The situation with strain XH56-701 was similar but somewhat more complicated due to the presence of the plasmid. At the permissive temperature of 30°. the ..L1.0-L7/L12 genes were transcribed about five-fold more frequently than the spc cluster of ribosomal protein genes because of the amplification in the gene copy number. Total transcription of the 3 gene (present on both the chromosome and the plasmid) and the 3' gene (present only on the chromosome) was elevated about four-fold compared to the non-plasmid parent. The bulk of this transcription was shown to be from the 3 gene by competition hybridization (data not shown; also see Reference 29 ); when this was taken into account it becomes apparent that the relative frequency of transcription of the 3 gene (values in parenthesis in Table VII) was elevated about six-fold compared to the non-plasmid strain and that the transcription attenuator in the vicinity of the L7/L12-3 intergenic space on the plasmids continue to terminate about 80% of the transcripts. When the plasmid strain was subjected to the partial restriction the relative transcription of the proximal L10-L7/L12 genes again increased about two-fold from 5.44 to 12.04. At the same time transcription of the distal 3 and 3' genes increased about five-fold from 0.67 to 3.48 and the imbalance in the synthesis of 3=3' mRNA remained about 6:1 (Table VII). Again this increase was due to both increased initiation of transcription at the promotor and a relaxation of transcription termination in the vicinity of the L7/L12-3 intergenic space. Transcription of the cluster 100 X H -56 "i 1 XH56-701 1 r 8 0 -Z o < 60 z LLI k-I— < V 4 0 -2 0 -0 J L J L 0 30 60 0 30 60 TIME (minutes after temperature shift) Figure 12: Transcription termination at the attenuator following partial restriction of RNA polymerase activity. The degree of transcription termination at the attenuator was estimated following the partial restriction of RNA polymerase activity from the relative frequency of transcription of the proximal L10-L7/L12 genes and the distal 3 and 3' genes (or 3 gene alone in strain XH56-701) as presented in Table VII, left panel, strain XH56; right panel, strain XH56-701. of ribosomal protein genes in the spc cluster remained about constant whereas transcription of the RNA polymerase a gene was elevated again about two-fold by the restriction. Synthesis of L7/L12, 3 and 3' Protein. By the combined utilization of the rpoC56 mutation to vary the level of transcription within the L10 operon and the plasmid pJC701 to amplify the copy number of the proximal portion of the operon including the rpoB gene, it was possible to achieve a ten-fold and a thirty-fold modulation in the transcription intensity of the proximal L10-L7/L12 genes and the distal 3 gene respectively. Obviously, it was of interest to know how these changes in the levels of the respective transcripts related to the synthesis rates of the corresponding proteins. To determine this cultures of XH56 and XH56-701 were grown at 30° and then shifted to 38.5°C. To measure the L7/L12 synthesis rate samples were removed prior to and at 3 various times after the temperature shift and pulse labeled with ( H)-14 leucine. The samples were mixed with ( C)leucine labeled carrier cells and extracts were prepared and electrophoresed in urea polyacrylamide 3 gels (Fig.13). The fraction of the total incorporated ( H)leucine radio-14 activity associated with L7/L12 was quantitated using the C radioactivity as an internal standard. The results indicate that in strain XH56 the relative synthesis rate of L7/L12 was enhanced only by about 20% during the 45 minutes of restriction compared to a two-fold increase in relative synthesis rate of the mRNA (Table VI-II).In the plasmid containing strain XH56-701 growing at 30°C the relative synthesis rate of L7/L12 protein was 1.26 fold greater than in the non-plasmid even though transcription of the mRNA was five-fold V B C 1 I B' B L 1 2 L 7 * a a a I f s -f j * 4 Figure 13: Autoradiograms of L7/L12 and 3 and 3' proteins separated by polyacrylamide gel elctrophoresis. Strains XH56 and XH56-701 were pulse labeled with radioactive amino acids prior to and at various times after the partial restriction of RNA polymerase activity. See Tables VIII and IX for details. The L7 and L12 ribosomal proteins were separated from the bulk of other proteins jji urea-polyacrylamide gels (A, autoradiogram illustrating the C carrier radioactivity). The 3 and 3' RNA polymerase proteins were separated from the bulk of other proteins on sodium dodecylsulfate-polyacrylamide gels: B, XH56 at 30°C (left); at 38.5°C, 15 min (center); and at 38.5°C, 45 min (right); C, XH56-701 at 30°C (left); at 38.5°C, 15 min (center); and at 38.5°C, 45 min (right). Table VIII; Relative synthesis rate of L7/L12 ribosomal protein Samples of cultures(5 ml) growing at 30°C or shifted to 38.5°C 3 were exposed to ( H)leucine (specific activity 53 Ci/mmol; 20 yCi/ml) for 5 min. Incorporation was terminated by the addition of nonradio-active leucine (50 yg/ml) and isoleucine (50 yg/ml) and the samples were further incubated for 2.5 min. The cells were harvested by 14 centrifugation and mixed with C labeled carrier cells (25 ml) of o 14 XH56/701 grown at 30 C and exposed to ( C)leucine (specific activity 342 mCi/mmol; 2 yCi/ml) for two generations. Following centrifugation, the samples were resuspended in 0.3 ml of 0.05 M Tris and 0.01 M EDTA, pH 8.1, and disrupted by freeze thaw treatment in the presence of 10 yg of lysozyme (23 ) followed by sonication for two 10 sec periods. Following acetic acid extraction (23) ribosomal proteins were dialyzed for 36 hr against 8 M urea and 0.05 M 3-mercaptoethanol. Proteins were electrophoresed on 1.5 mm thick, 4% polyacrylamide slab gels in the presence of 8 M urea at 40 mA for 4-5 hr ( 29,78 ). The gels were fixed in 1 N HCl, washed with 7% acetic acid and dried. The radioactive bands corresponding to the L7 and L12 proteins were cut from the dried gels and oxidized with 0.3 ml of 40% "i"n t*ie presence of 2% NH^OH for 18 hr at 37°C. Residual H ^ was inactivated by the addition of 0.75 ml of catalase (4 mg/ml in 10 mM Tris, pH 7.4) 3 14 and the H and C radioactivity was determined by scintillation counting. The relative differential synthesis rate of L7/L12 was calculated as f L 1 2 = ( 3H/ 1 4C) in L7 + L12/(3H/14C) in total protein and normalized to a value of 1.0 for the XH56 30°C control sample. The differences in the isotope ratio of L7 and L12 simply reflect minor variations in the acetylation of the protein during the labeling intervals but in all cases the L7 form predominates by about 4:1. Minimum amounts of radioactivity used for ratio calculation were 800 and 3000 cpm for L12 and L7 proteins, respectively. Table VIII: Relative synthesis rate of L7/L12 ribosomal protein Strain 3,, ,14„ _ ^ _ ^. H/ C Isotope Ratios Total L7 L12 L7 + L12 ^7/^12 Protein XH56 30° 0.64 0.51 0.84 0.56 0.87 (1.00) 38.5°-12.5' 0.56 0.45 1.03 0.53" 0.94 (1.08) 38.5°-45' 0.57 0.51 1.27 0.60 1.05 (1.21) XH56-701 30° 1.04 1.05 1.67 1.14 1.10 (1.26) 38.5°-12.5' 0.90 1.12 1.36 1.15 1.28 (1.47) 38.5°-45' 0.58 0.88 1.44 0.96 1.64 (1.89) elevated. The restriction further increased transcription to more than ten-fold above the level in the non-plasmid strain at 30°C but increased the synthesis rate of L7/L12 protein to a value of only 1.89. Thus the increased transcription of the L7/L12 gene caused by either amplification of the gene copy number or the partial restriction of RNA polymerase activity was generally ineffective in stimulating the synthesis rate of L7/L12 protein. The relative synthesis rates of the 3 and 3' subunits of RNA poly-merase were also measured in the respective cultures prior to and following the temperature restrictions (Fig. 13, and Table IX).. In the non-plasmid strain the restriction resulted in about a 3.5 fold increase in subunit synthesis rates and was comparable to the increase in transcription of 3 and 3' mRNA during this period. In the plasmid strain labeled at 30°C the synthesis rate of 3 was elevated about 4-fold due to amplification of the rpoB gene whereas the synthesis rate of 3 mRNA was estimated to be nearly 6-fold above the control rate. Following the temperature re-striction the synthesis rates of the 3 and 3' subunits were both elevated about 3.8 fold whereas transcription of 3 and 3' mRNA sequences increased more than five-fold. Table IX: Relative synthesis rate of RNA polymerase 3 and 3' subunits. Strain 3 3' 3 + 3 ' XH56 30° 0.42 0.58 1.00 38.5°-12.5' 1.08 1.72 2.80 38.5°-45' 1.27 2.12 3.39 XH56-701 30° 1.77 0.70 2.47 38.5°-12.5' 3.48 1.53 5.01 38.5°-45' 6.86 2.62 9.48 Cellular proteins were pulse labeled at 30°C and following a shift to o 14 38.5 C by exposing 2.5 ml culture samples to ( C) leucine (specific activity 342 mCi/mmol; 2 yCi/ml) for 3 min. Incorporation was termin-ated by addition of nonradioactive leucine (50 yg/ml) and isoleucine (50 yg/ml) followed by a further 2.5 min incubation to allow for completion of nascent polypeptide chains. Lysates were prepared for SDS-polyacrylamide gel electrophoresis as described previously (26 ). The radioactive bands corresponding to the 3 and proteins were cut from the gels, oxidized with peroxide and the C radioactivity was determined. The fraction of the total radioactivity associated with the 3 and 3' peptides was calculated and normalized to 1.0 for 3 + 3' in the XH56, 30 C control sample. The minimum amounts of radioactivity in the individual proteins was greater than 150 cpm for the 30 C samples and 500 cpm for the restriction samples. DISCUSSION The regulation of ribosomal protein and RNA polymerase genes is exceedingly complex and interrelated. In the L10 transcription unit the major promotor and a transcription attenuator have been located about 350 nucleotides in front of the L10 structural gene and in the L7/L12-3 intergenic space respectively (5,112,142). In addition, evidence for several minor promotors located in the Ll0-L7/L12and the L7/L12-3 intergenic spaces have been obtained (5,38,84,103 ). In interpreting our results we have tended to ignore these secondary promotors since their physiological significance remains obscure and their levels of activity are apparently very low. It has been further assumed that the increases observed in the transcription of the proximal L10-L7/L12 genes (see Table VII) resulted from increased initiation at the major L10 promotor. It is possible, however, that transcription of the proximal genes is regulated not by initiation but rather by termination at an attenuator site within the leader region of the operon. Attempts to identify a 5' terminal RNA fragment produced by termination of the trans-cripts initiated at P^iq have so far been unsuccessful in these strains (unpublished results). The reduction in the transcriptional capacity caused by the partial temperature inactivation of core RNA polymerase selectively stimulates the transcription of genetic units which contain RNA polymerase component genes. In the L10 transcription unit the frequency of transcription initiation (estimated as the transcription of the promotor proximal L10 and L7/L12 ribosomal protein genes) was enhanced about two-fold whereas transcription of the distal 3 and 3' RNA polymerase genes was enhanced nearly four-fold. Thus reduced transcriptional capacity selectively stimulates transcription initiation at the major L10 promotor and reduces termination at the L7/L12-3 attenuator. Alternatively, activation of secondary low level promotors within the transcription unit might also be involved. These observations substantiate and extend earlier experi-ments which employed less specific probes for the RNA-DNA hybridization (85 ). Transcription of the a subunit gene located in the major ribosomal protein gene cluster near 72 min was also selectively stimulated about two-fold by the restriction; the general level of transcription of the ribosomal protein genes of this cluster was enhanced less than 25%. This implies that the small stimulation in the general transcription of this region of the chromosome caused by the partial inactivation of RNA polymerase was specific and confined predominantly to the ot transcription unit rather than units containing only ribosomal protein genes. The presence of the ColEl plasmid pJC701 in strain XH56 amplifies the copy number of the proximal portion of the L10 transcription unit without complementing the temperature sensitive chromosomal rpoC56 mutation. During a restriction the partial L10 transcription units located on the plasmid responded in a manner analogous to the chromosomal unit by in-creasing at the L7/L12-3 attenuator. By combined use of the temperature restriction and the plasmid mediated amplification, it was possible to obtain a 10 and 30 fold variation in the transcription intensity of the L10-L7/L12 genes and 3 gene respectively. The relative synthesis rates of L7/L12 and 3 subunit proteins were varied, however, by only 2 and 15 fold respectively; this indicates that the translation efficiencies of the respective mRNA sequences were reduced to 20% and 50% of the normal level. Fallon ^t al.( 37 ) have suggested that free ribosomal proteins influence the stability of their own mRNAs and thus regulate their own synthesis rates. Our measurements, made under conditions of plasmid mediated gene amplification, indicate that although L10-L7/L12 mRNA sequences are somewhat less stable than normal they still accumulate in excess of the synthesis rate of L7/L12 protein ( 29,39,40,41 ). This suggests that translation is restricted possibly through the action of L7/L12 and/or L10 protein either directly by inhibition of initiation complex formation or indirectly by mRNA inactivation through modification or abnormal processing. The failure to load the mRNA with translating ribosomes may contribute to its reduced half life. In any event, the overproduction of L7/L12 protein is severely curtailed during an RNA polymerase restriction. This presumably also involves L10 synthesis, occurs subsequent to the synthesis of L10-L7/L12 mRNA sequences and appears to require the small excess production of L7/L12 protein (39,41). In related experiments which support these views Fiii et al. recently characterized a series of mutants which are defective in translation of L10 mRNA sequences. These point mutations are localized in the leader region about midway between P^io anc* t'ie structural gene in a region of dyad symmetry, and do not appreciably affect transcription of the L10 structural gene. It was suggested that these mutations alter the con-formation of the mRNA transcript and thus mimic the normal translation inhibition effect achieved by the interaction between some regulatory element and the wildtype mRNA sequences (40 ). It has been further suggested that the genes specifying the 3 and 3' subunits of RNA polymerase are autogenously regulated by free subunits or intermediates in the assembly pathway (121). In fact, the intermediate 0^3 as well as other possible intermediates apparently inhibit the syn-thesis of 3 peptide in vivo and in vitro (44,130,144) • It is possible that this inhibition is post transcriptional in nature and in general terms analogous to the mechanism which restricts the overproduction of L7/L12 from excess mRNA. During the restrictions reported here 3 mRNA in strain XH56-701 is translated with an efficiency of about 50% and the intermediate 0^3 could easily be accumulating. Independent support for this post trans-criptional regulation of 3 subunit synthesis by RNA polymerase assembly intermediates has recently been presented (70 ). LITERATURE CITED 1. Adhya, S., and M. Gottesman (1978). Ann. Rev. Biochem. 47: 967-996. 2. Allet, B., R. Roberts, R. Gesteland and R. Solem (1974). 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