@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix dc: . @prefix skos: . vivo:departmentOrSchool "Medicine, Faculty of"@en, "Biochemistry and Molecular Biology, Department of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Briant, Douglas James"@en ; dcterms:issued "2009-11-16T23:57:03Z"@en, "2003"@en ; vivo:relatedDegree "Doctor of Philosophy - PhD"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description """Ribonucleic acid (RNA) is a vital molecule in the cell. Messenger RNA (mRNA) serves as the intermediate between DNA and protein while ribosomal RNA (rRNA) and transfer RNA (tRNA) catalyze translation. RNA is processed and ultimately degraded by ribonucleases. The majority of the endoribonucleolytic activity in Escherichia coli is derived from RNase E. This 1061 amino acid protein associates with at least three other proteins to form a complex called the RNA degradosome. This work established that the degradosome does not assemble de novo with successive rounds of degradation. It also determined that RNase E lacks 5'-phosphatase activity. Studies into the activity of RNase E are confounded by the fact that it associates into a complex with other RNA processing enzymes. We therefore utilized RNase G, which shares 35% amino acid sequence identity (50% similarity) to the catalytic domain of RNase E, as a model for RNase E. RNase G is the endonuclease responsible for forming the mature 5'-end of 16S rRNA. Non-denaturing purifications for RNase G were developed and the correct Nterminal sequence of the protein unambiguously identified. Through cross-linking studies, sucrose gradient centrifugation and gel filtration, it was determined that RNase G, and by inference, RNase E, exists primarily as a dimer. Site-directed mutagenesis was utilized to elucidate the role of six cysteine residues, including two highly conserved cysteines, of RNase G. None of the mutations resulted in a loss of activity, although subtle influences on structure and activity were observed with the RNase G variants. The S1 domain, which potentially binds RNA, was deleted without inactivating the enzyme. Further studies are required to determine if the S1 domain plays a role in substrate recognition. Finally, examinations using synthetic, chimeric RNA-DNA oligonucleotides revealed the chemical requirements for recognition and cleavage of a substrate by RNase E or RNase G. I concluded that a single 2'- OH group 5' to the site of cleavage was sufficient for endoribonucleic activity. This work established RNase G as a model for investigating the activity and structure of the catalytic domain of RNase E."""@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/15021?expand=metadata"@en ; dcterms:extent "13035845 bytes"@en ; dc:format "application/pdf"@en ; skos:note "FUNCTIONS AND PROPERTIES OF RNase G and RNase E FROM Escherichia coli. by Douglas James Briant Hon. B. Sc., The University of Waterloo, 1993 M. Sc., The University of Waterloo, 1995 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Biochemistry and Molecular Biology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July, 2003 © Douglas James Briant, 2003 UBC Rare Books and Special Collections - Thesis Authorisation Form Page 1 of 1 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r 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 f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of 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 granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of BiflCKf nAvCT 0.1 mM DTT, 0.2 mM PMSF. Buffer D2- 100 mM Tris-HCl pH 7.5, 10% glycerol, 68 mM NH4CI, 0.2 mM EDTA, 1 mM MgCI2, 0.1 mM DTT, 0.2 mM PMSF. Buffer D3- 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 20% glycerol. Tris/Boric acid/EDTA (TBE) buffer- 90 mM Tris, 90 mM boric acid, 2 mM EDTA PTBN- 20 mM Na-phosphate pH 7.0, 0.05% Tween 20, 0.1 mM bovine serum albumin, 0.85% NaCl, 1 mM NaN3. Phosphate Buffered Saline (PBS)- 137 mM NaCl, 2.7 mM KCI, 10 mM Na2HP04, 1.8 mM KH2P04, pH to 7.4 with HCI. 2.4. Polymerase chain reaction DNA was amplified using the polymerase chain reaction (Mullis etai., 1986). For site-directed mutations, the Stratagene QuikChange kit, which employs PfuTurbo DNA polymerase. Taq DNA polymerase (Life Technologies) was used for all other applications. The manufacturers' protocols were followed for both 41 enzymes, with incubations performed in a Perkin Elmer GeneAmp 2400 thermocycler. 2.5. Other molecular biological methods Standard molecular biology techniques, including restriction endonuclease treatments, ligation, subcloning, agarose gel electrophoresis and generation of RNase-free water were performed as previously described, unless otherwise noted (Sambrook, 1989). 2.6. Enzyme purifications Note: for all purifications, the ratio of protein of interest to contaminant was determined by SDS-polyacrylamide gel electrophoresis, which is described in Section 2.6.1. 2.6.1. Untagged RNase G BL21 (DE3) transformed with pDB6 (strain DB6) resulting in strain DB6. DB6 was grown at 37° in 600 ml LB with ampicillin (100 ug/ml) to an A6oo of 0.4 - 0.6 and then for approximately 16 hours at 20° with 0.1 mM IPTG. Cells were harvested by centrifugation and resuspended in 5 ml Buffer II (section 2.3), with aprotinin, leupeptin, pepstatin A (all to a final concentration of 0.02 ug/ml). Cells were lysed at 12,000 psi in a French Pressure cell (Aminco) and one additional volume of Buffer II was added. The diluted lysate was incubated on ice for 20 min with1.5 ug/ul DNase I. Lysates were cleared by centrifugation at 30,000 x g 42 to form an S30 (Fraction 2) and a pellet (Fraction 1), which was resuspended in one volume (this and subsequent volumes are relative to the S30) of Buffer II. RNase G was precipitated from the S30 (Fraction 2) by the addition of 26% w/v ammonium sulphate followed by centrifugation at 17,500 x g. Precipitated proteins were taken up in approximately 10 volumes of Buffer II (to give Fraction 3) and loaded onto a 25 ml Heparin-agarose column. Proteins were eluted with a gradient of 50 mM to 2 M NaCl in Buffer II. Fractions containing RNase G were pooled and diluted 1:1 with 25 mM Tris-HCl pH 7.6, 5 mM DTT (forming Fraction 4) prior to loading on a 1 ml Mono Q column (Pharmacia). Proteins were eluted with a gradient (25 mM Tris-HCl pH 7.6, 5 mM DTT, 50 mM - 2 M NaCl). Fractions containing RNase G were pooled (Fraction 5), divided into aliquots, frozen in liquid N2 and stored at -70°. Protein was quantified by staining with SYPRO Red (Pharmacia) and analysis on ImageQuant software (Molecular Dynamics). 2.6.2. RNase G mutants Cysteine to serine mutations were introduced into C-terminally His-tagged RNase G using the PCR-based Stratagene QuickChange kit. The six cysteines of RNase G are located at residues 79, 162, 402, 405, 408 and 421 (Fig. 3.1). Templates and primers used for mutagenesis are listed in Table 2.2. Plasmids designated pDB were constructed by myself, while those designated pJH were made by Janet S. Hankins. To perform the mutagenesis, 0.1-1 ng/ul template DNA, 25 ng/ul each of forward and reverse primers, 0.3 ng/ul each of ATP, CTP, 43 Table 2.2. Primers and templates used for construction of RNase G clones Plasmid Mutation Template Primer Sequence pDB6 wild-type chromosomal DNA For-CCGTAGTCGGATCCCCGCTGGTTG Rev-CTCAAAAACCCGGATCCGGATGGCGG pDB1 C-terminal His chromosomal For-CCGTAGTCGGATCCCCGCTGGTTG tag DNA Rev-CGCGGATCCTCGAGCATCATTACGACGTCAAACTGC pDB4 AS1 (A36-135) pDB1 For-CATATTGAACGTGAGGCGCGACGCGCTTCTCACGTTGGGGTTT c c Rev-GGAAACCCCAACGTGAGAAGCGCGTCGCGCCTCACGTTCAAT ATG pJH1b C421S pDB1 For-CCGTGGAAACGGTAAGCTATGAAATCATGCGCGAG Rev-CTCGCGCATGATTTCATAGCTTACCGTTTCCACGG pJH4 C402S pDB1 For-GCACGTACTGTCTAACGAATGCCC Rev-GGGCATTCGTTAGACAGTACGTGC pJH5 C162S pDB1 For-CGCAGAGTATTCCGAACGAGCAGGG Rev-CCCTGCTCGTCGGAATACTCTGCG pDB5 C402S, C405S pDB1 For-CTGTGTAACGAATCCCCAACCTCCCACGGTCGCGG Rev-CCGCGACCGTGGGAGGTTGGGGATTCGTTACACAG pJH3 C402S, C405S,pDB5 For-GCACGTACTGTCTAACGAAATCCC C421S Rev-GGGATTCGTTAGACAGTACGTGC 44 GTP and CTP, were mixed with 0.05 U/ul PfuTurbo DNA polymerase (Stratagene) in 10 mM KCI, 10 mM (NH4)2S04, 20 mM Tris-HCl pH 8.8, 2 mM MgS04, 0.1% Triton X-100 and 0.1 ug/ul bovine serum albumin. As directed by the manufacturer's instructions, 18 cycles of PCR were performed on a Perkin Elmer GeneAmp 2400. Products of PCR (5 pi each in 0.5 pi 50% glycerol, 50 mM EDTA, 0.25% bromophenol blue, 0.25% xylene cyanol) were analyzed on a 0.8% agarose gel in TBE buffer (section 2.3). Successfully amplified products were treated with 5U Dpnl (Stratagene) at 37°C for 1 hr to remove template DNA. The mutant plasmids were then transformed into Gibco Subcloning Efficiency DH5a Chemically Competent Cells, following the manufacturer's instructions. Plasmids were isolated from DH5a clones, and the mutation confirmed by sequencing or restriction enzyme analysis. The desired plasmids were transformed into BL21(DE3) cells, to allow protein overexpression. RNase G from mutant strains was purified and quantified as described for wild-type His-tagged RNase G (Section 2.6.3). 2.6.3. His-tagged RNase G Plasmid pDB1 was transformed into E. coli BL21(DE3) to form strain DB1. Purification of His-tagged RNase G was similar to the method of Jiang, et al (Jiang et al., 2000). In outline, 2 I cultures of DB1 were grown at 37°C in LB containing 75 u.g/ml kanamycin to an A6oo of 0.4 - 0.6 and then overnight at 20°C with 0.1 mM IPTG. Cells were harvested at 3,000 x g, resuspended in 10 ml binding buffer (25 mM Tris-HCl pH 7.6, 500 mM NaCl, 7 mM p-mercaptoethanol) 45 with 0.5 mM PMSF, and lysed at 12,000 psi in a French Press. Lysates were diluted with one additional volume of binding buffer and treated with 1.5 ug/ul DNase I. Insoluble material was removed by centrifugation at 30,000 x g and the resulting supernatant (S30) was diluted with one volume binding buffer. The dilute S30 was mixed with an appropriate volume (2 to 2.5 ml) of Talon resin (Clonetech) in a 50 ml screw-cap tube (Falcon) for 30 min at 4°C on a Nutator orbital shaker (Rose Scientific). Following binding, the resin was poured into a 10 ml column and washed with 3 volumes of binding buffer containing 5 mM imidazole. Proteins were eluted in three steps with four column volumes each of Buffer I (section 2.3) containing 10 mM, 50 mM or 500 mM imidazole. Elution fractions 2 and 3 (50 mM and 500 mM imidazole respectively) were pooled and loaded onto a 15 ml SOURCE Q column (Pharmacia) in Buffer I. RNase G was eluted with a 50 mM - 2 M NaCl gradient in the same buffer. Fractions containing the highest ratio of RNase G to contaminants were pooled, aliquots were frozen in liquid N2, and stored at -70°C. Protein preparations were quantified by staining with SYPRO Red (Pharmacia) and analysis on ImageQuant software (Molecular Dynamics). 2.6.4. Purification ofpnp13 degradosomes Purification of pnp13 degradosomes has been previously described (Coburn et al., 1999). E. coli strain RD100 (constructed by R. P. Dottin, a gift from M. L. Pearson) was grown overnight by the Fermentation Pilot Plant Facility, Biotechnology Laboratory, University of British Columbia. The resulting cell 46 pellet (approximately 25 g) was resuspended in 50 ml Buffer IV (section 2.3) with 3 mM EDTA, 1.5 mg/ml lysozyme (Sigma), and incubated on ice for 70 min with intermittent stirring. An additional 20 ml Buffer IV with 30 mM Mg-acetate, 3% Triton X-100 and 20 ug/ml DNase I (Sigma) were added, followed by 4° C incubation on ice. NH4CI was added to a final concentration of 1M and the sample was centrifuged for at 4°C for 60 min at 30,000 x g. The resulting supernatant (S30) was centrifuged at 200,000 x g at 4°C for 2 hr in a Ti 60 rotor (Beckmann). Degradosomes were precipitated from the supernatant (S200) by addition of 26% w/v ammonium sulphate with stirring on ice for 40 min and centrifugation at 10,000 x g at 4°C for 1 hr. The AS26 pellet was resuspended in Buffer V (section 2.3). The preparation was loaded onto a 25 ml SP-Sepharose FF column (BioRad) followed by two stepwise washes with four column volumes each of Buffer V with 50 mM NaCl or 300 mM NaCl. Proteins were eluted with Buffer V containing 1% genepol-X80 and 1 M NaCl and fractions collected. Fractions with the highest ratio of degradosomal proteins to contaminants (as determined by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Brilliant Blue R-250) were pooled and loaded onto a 10 ml Affi-Blue gel column (BioRad). The column was washed with 10 volumes of Buffer V with 50 mM KCI. Proteins were eluted in Buffer V with a 300 mM to 3 M KCI gradient, and fractions collected. Fractions with the highest ratio of degradsosomal proteins to contaminants were pooled, and concentrated by precipitation with 26% ammonium sulphate, as previously described. The pellet was taken up in 1 ml Buffer V with 50 mM NaCl and loaded onto a 100 ml Bio-Gel A5m column (BioRad). Fractions with the highest ratio of degradosomal proteins to 47 contaminants were pooled and concentrated to approximately 0.5 mg/ml in a 15 ml Ultrafree Biomax-5K centrifugal filter device (Millipore). 2.6.5. Electropurification of RNase E Electroelution of RNase E was performed as previously described (Cormack et al., 1993) with some modifications. Briefly, 500 ml of M9ZB containing 50 ug/ul ampicillin was inoculated with strain GM402, which expresses full-length RNase E under the control of a T7 promoter. The culture was grown to an A6oo of 0.4, then induced with 1 mM IPTG and shaken at 30° for 5 hrs. Cells were pelleted by centrifugation, resuspended in 5 ml Buffer II (section 2.3), with 7.5 % glycerol and aprotinin, leupeptin, pepstatin A (all to a final concentration of 0.02 u.g/ml) and lysed a French Pressure cell (Aminco) at 8,000 psi. The lysate was incubated on ice for 10 min with1.5 pg/ul DNase I. Lysates were cleared by centrifugation at 30,000 x g to form an S30. Three additional volumes of Buffer II with 5% glycerol were added to the S30 and RNase E was precipitated by the addition of 26% w/v ammonium sulphate followed by centrifugation at 17,500 x g. The pellet was redissolved in Buffer II with 5% glycerol, 0.02 ug/ml leupeptin, and dialzyed in Spectra/Por dialysis tubing (MWCO 12,000 - 14,000; Spectrum Laboratories) at 4° for 1 hr in 400 mis Buffer D1 (section 2.3). The RNase E sample was subsequently dialyzed for 2 hrs in 800 mis Buffer D2 (section 2.3). Following dialysis, one volume of 2 x SDS-sample buffer was added to each sample (section 2.7.1) which was then loaded into a single well, the width ofthe gel, and subjected to electrophoresis on a 6% SDS-polyacrylamide gel (49:1 48 acrylamide:bis-acrylamide) (modified from 10% SDS-polyacrylamide gel, described in section 2.6.1). Following electrophoresis, slices were removed from each side of the gel and stained with Coomassie Brilliant Blue R-250 to visualize the location of the RNase E protein band. The band was then excised from the gel, placed in dialysis tubing (Spectra/Por, MWCO 12,000 - 14,000; Spectrum Laboratories) with a minimal volume of Laemmli buffer (section 2.7.1) and electroeluted at 100 volts for 7 hrs in a horizontal electrophoresis unit containing Laemmli buffer. Following electroelution, the liquid (approximately 2 ml) was removed and protein precipitated overnight at -20° in 5 volumes of acetone with 0.2 mM DTT. Protein was recovered by centrifugation followed by three 10 ml washes with 80% acetone, 0.2 mM DTT. The RNase E pellet was dissolved in 350 ul Buffer D3 (section 2.3) with 6 M guanidine hydrochloride and diluted with 5 ml Buffer D3. The sample was then dialyzed in two 400 ml changes of Buffer D1 at 4° for 6 and 4 hours, respectively. The sample was then concentrated to approximately 300 pi in a 4 ml Ultrafree Biomax-5K centrifugal filter device (Millipore). 2.7. Protein analysis 2.7.1. SDS-polyacrylamide gel electrophoresis One volume of 2 x SDS-sample buffer (120 mM Tris-HCl pH 6.8, 3% sodium dodecylsulphate (SDS), 50 mM DTT, 10% glycerol, 0.1% bromophenol blue) was added to an appropriate volume of protein sample (generally 4-7 pi) and the mixture boiled for 2 min. Samples were loaded onto a 10% polyacrylamide gel (36:1 acrylamide:bis-acrylamide) containing 1% SDS and separated at 150-49 200V in Laemmli buffer (25 mM Tris-HCl, 192 mM glycine, 0.1% SDS)(Laemmli, 1970) along with Broad Range SDS-page standards (BioRad). Proteins were visualized by staining either with Coomassie Brilliant Blue (0.5 mg/ml Coomassie Brilliant Blue R-250 (BioRad), 45% methanol, 10% acetic acid) followed by destaining with 5% acetic acid, 5% ethanol, or by staining with SYPRO Red (diluted 1:5000 in 7.5% as recommended by the manufacturer; Pharmacia) followed by a rinse with distilled water and imaging on a Typhoon 8600 imager (Molecular Dynamics). 2.7.2. Western blotting for RNase G Proteins were separated by SDS-polyacrylamide gel electrophoresis (section 2.7.1). Gels were not stained following electrophoresis, but were instead blotted to Trans-Blot Transfer Medium nitrocellulose paper (BioRad) at 250 mA for 2 hrs in transfer buffer (3 mM Na2C03, 10 mM NaHC03, 20% methanol; Dunn, 1986). Blots were shaken at room temperature for 1 hr in PTBN (Section 2.3) with 5% casein. Blots were subsequently incubated with shaking in PTBN with 5% casein and the primary antibody, rabbit anti-RNase G (gift from Janet S. Hankins). Blots were exposed to three 5 min washes with PBS followed by shaking for 45 minutes in PBS containing the secondary antibody (goat anti-rabbit; Amersham, 1:3000 dilution). Washes with PBS were repeated and bands visualized by the addition of ECL (Pharmacia) chemiluminescent reagent and exposure to x-ray film. 50 2.7.3. N-terminal protein sequencing A sample of wild type RNase G was subjected to SDS-polyacrylamide gel electrophoresis and blotted as described in section 2.7.2, with the exception that proteins were blotted onto Immobilon-P PVDF membrane (Millipore). Following blotting, the membrane was washed 3 times with H2O, and stained for 2 min in Coomassie R-250 Brilliant Blue (see section 2.7.1). The blot was destained in several changes of 50% methanol, 10% acetic acid, and allowed to air dry. Bands of interest were excised, and gas phase sequencing was performed at the University of Victoria-Genome B. C. Proteomics Centre. 2.7.4. Mass spectrometry 50 pi samples of protein (0.5-1 pg/ul) were analyzed using electrospray mass spectrometry by Dr. Shuming Hu at the U. B. C. Laboratory of Molecular Biophysics. 2.7.5. Protein cross-linking experiments Cross-linking was performed as previously described (Klingenberg and Appel, 1989). Briefly, RNase G (0.08-0.16 ug/uJ) was incubated at 4° for 25 minutes with 12.5-30 pM CuS04 and 0.65 mM 1,10-phenanthroline in 30 mM Na2S04, 0.1% Triton X-100, 0.1 mM EDTA, 5 mM Tris-HCl pH 6.8. Reactions were quenched by addition of 2 mM N-ethylmaleimide, 15 mM EDTA followed by incubation at 25°. Cross-linked protein samples were separated on a 7.5% SDS-polyacrylamide gel (Laemmli, 1970) and visualized by staining with Coomassie 51 Brilliant Blue R-250. For 2-D analysis, sample lanes were sliced from the gel and soaked in either non-reducing sample buffer or reducing sample buffer (sample buffer +100 mM DTT) for twenty minutes at ambient temperature. This gel slice was then placed horizontally on top of a second 7.5% gel and subjected to electrophoresis. Proteins were visualized with Coomassie Brilliant Blue R-250. 2.7.6. Circular dichroism Purified RNase G was diluted to 4.6 pM (0.26 pg/pl) in 25 mM Tris-HCl pH 7.6, 300 mM NaCl, and 400 pi was loaded into a 0.2 mm quartz cuvette (Hellma). Wavelength scanning from 190 nm to 300 nm in a Jasco J-810 Spectropolarimeter (Jasco) was performed on each sample. Maximum CD values occurred at approximately 218 nm, so this wavelength was chosen for the CD melting studies. Sample temperature was increased from 20°C to 70°C at 1°C per minute. The melting temperature for each protein corresponded to the inflection point in the CD scan, and was calculated using Spectra Manager software (Jasco). 2.7.7. Sucrose gradient centrifugation 2 ml gradients were poured in 11 x 34 mm Ultra-Clear tubes (Beckmann) in a stepwise fashion by layering 500 pi each of 20%, 15%, 10% and 5% w/v sucrose in 25 mM Tris-HCl pH 7.6, 300 mM NaCl, 10 mM DTT, followed by incubation at room temperature for 1 hr, as previously described (Loewen and Molday, 2000). Purified RNase G (30 ug) and 4 pg each of bovine serum albumin and aldolase standards (Pharmacia) were layered on top ofthe gradient 52 and the samples were centrifuged at 4°C for 6 hours at 50,000 rpm in a Beckmann TLS-55 rotor. Four drop fractions were collected by puncturing the bottom of the tube. 6 pi portions of each fraction were mixed with SDS-sample buffer (section 2.7.1) and products were separated on a 10% SDS-polyacrylamide gel. Proteins were visualized by staining with SYPRO Red (Pharmacia) and quantified using ImageQuant software (Molecular Dynamics). 2.7.8. Gel filtration size determination A Superdex S200 column was calibrated by measuring the elution volume of thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), BSA (67 kDa), ovalbumin (45 kDa) and chymotrypsin (25 kDa)(all standards purchased from Pharmacia) on an Akta Explorer (Pharmacia), with the elution volumes determined using Unicorn software (Pharmacia). Samples were run in 25 mM Tris-HCl pH 7.6, 320 mM NaCl. A standard curve was constructed from this data. Samples (amounts listed in the figure legend) were loaded and passed through the column in Buffer I (section 2.3). The elution volume analyzed using Unicorn software (Pharmacia). Sizes of the samples were extrapolated from the standard curve. Calibration of the column was performed by R. Pfeutzner. 2.8. RNA substrates and endoribonuclease assays 2.8.1. Full-length substrates Full-length RNA substrates were prepared as previously described (Cormack and Mackie, 1992; Mackie, 1998). Briefly, plasmid DNA (see Fig. 2.1 for 53 Figure 2.1. Templates for runoff RNA transcription. The transcription reaction is described in section 2.8.1. Panel a depicts the template for 9S RNA, which is transcribed from an SP6 promoter at the +1 site and extends the full length of the gene to residue 246 corresponding to a cleavage by >Acc/ or Hindi. In panel b, the coding region for S20 is indicated as an open box. Transcription from an SP6 promoter begins at the native P2 promoter, and the template is linearized with Dral at nucleotide 447, which corresponds to the natural termination 3' to the rho-independent terminator (see also Fig. 3.11b). Panel c shows the rrnB 16S rRNA cistron. The two native promoters (P1 and P2) are indicated with arrows, and the mature rRNA sequence is represented with and open box. Panel d represents the template derived from plasmid pGM119, which can be linearized with either BstUI or BamHI to yield a truncated pre-16S rRNA. Processing sites are indicated as follows: RNase III at residue -115, III; RNase E at residue -66, E; RNase G at the mature 5'-end, +1, G. The circle represents the approximate binding site of ribosomal protein S20. The horizontal line indicates the annealing site of oligo GMV 2011, which was used to direct RNase H cleavage at the RNase III processing site. Panel e shows the template derived from plasmid pGM122, which uses a T7 promoter to initiate transcription at the P2 site (see panel c). RNA transcripts corresponding to those transcribed from pGM122 could also be derived from cleaving substrates transcribed from pGM119 cleaved by RNase H directed by oligo GMV 2011, described above. 54 +1 S P 6 ^ r E _2_ Accl/Hincll +246 9SRNA | T E _2_ PJG9S.2 246 nt +92 ••\"«> E SPb S20 f r p s r ; 372 nt Dral +447 _L. T PGM79 * SH P1 P2 rrnB 16S rRNA cistron ilL E • A -115 -66 +1 576 nt 728 nt \"1 f BstUI BamHI +404 +556 ->| PGM119 >l 671 nt 55 templates) was linearized with an appropriate endonuclease. Template DNA (0.6 pg) was combined with 500 pM each of ATP, GTP and UTP, 100 pM CTP, 30 pCi a32P-CTP, 0.8 U/pl RNA guard, 6 U/pl SP6 or T7 RNA polymerase (depending on the template promoter) in 40 mM Tris-HCl pH 7.9, 6 mM MgCb, 2 mM spermidine, 10 mM NaCl and incubated for one hour at 37°C. The reaction was quenched by dilution and addition of NH4-actetate to 2M, EDTA to 5 mM. RNA was extracted with phenol/chloroform/isoamly alcohol (20:19:1) and precipitated overnight at -20°C with four volumes of ethanol. RNA was quantified by determining the ratio of total to TCA-precipitable scintillation counts (Sambrook, 1989). Following quantification, half the product (5'-triphosphorylated RNA) was diluted to a final concentration of 0.2 pmol/uJ in H20. The remainder was dephosphorylated in Buffer III (section 2.3) with 0.02 U/pl calf intestinal alkaline phosphatase. The phosphatase was inactivated byincubation in 8 ng/pl proteinase K in 0.1% SDS for 10 min at room temperature. This reaction was quenched with EDTA (9 mM) and the RNA was extracted with phenol/chloroform/isoamyl alcohol (20:19:1) and precipitated with ethanol. The dephosphorylated RNA was resuspended in Buffer III and rephosphorylated at its 5'-end by addition of 0.3 U/uJ polynucleotide kinase in the presence of 0.9 mM ATP and 0.8 U/uJ RNAguard (Amersham-Pharmacia). Samples of mono- and triphosphorylated RNA were separated simultaneously on a 6% urea-polyacrylamide gel for quantitation. 56 2.8.2. 5'-end labelling of oligonucleotides 200 pmoles oligonucleotide were incubated at 37°C for 30 mins in 20 ul Buffer III (section 2.3) with 0.5 U/ul polynucleotide kinase (Fermentas), 25 pCi y32P-ATP and 10 pmol/pl ATP. For unlabelled oligonucleotides, y32P-ATP was omitted and the concentration of ATP was increased to 60 pmol/pl. The reaction was quenched with 160 mM NH4-acetate, 8 mM EDTA and the reaction volume brought up to 50 pi with DEPC-treated water. The kinase was inactivated by heating the mixture at 65°C for 10 mins, according to the manufacturer's instructions. To precipitate oligonucleotides, the reaction volume was increased to 100 pi with 300 mM Na-acetate, 0.5 pg/pl yeast RNA instead of quenching the reaction. Residual proteins were extracted with phenol/chloroform/isoamyl alcohol (20:19:1) and the oligonucleotides were precipitated overnight at -20°C with 4 volumes of ethanol. The pellet was taken up in an appropriate volume of DEPC-treated water. 2.8.3. Standard endoribonuclease assays Labelled RNA (amounts indicated in Figure Legends) was denatured by heating in RNase Assay Buffer (section 2.3) for 2 min at 50°C, for 10 min at 37°C, and chilling on ice. Processing was initiated by the addition of purified RNase G, RNase E or purified degradosomes, with amounts of enzyme listed in the Figure Legends for each experiment, in a final volume of 30pl (Coburn et al., 1999). Incubation was continued at 30°C. Samples of 4 pi were withdrawn at appropriate times, quenched with 12 pi 90% formamide, 22 mM Tris, 22 mM boric acid, 0.5 mM EDTA, 0.1% each of bromophenol blue and xylene cyanol, 57 heat denatured, and separated on polyacrylamide gels containing 8 M urea, and separated in TBE buffer (Section 2.3). For assays using oligonucleotide substrates, products were analyzed on 15% polyacrylamide gels while larger RNAs, unless otherwise indicated, were separated on 8% polyacrylamide gels. Products were visualized by phosphorimaging. 2.8.4. Ribonucleoprotein assays Substrates based on pre-16S rRNA are depicted in Fig. 2.1. In each assay, unless otherwise indictated in the Figure Legend, 0.6 pmoles monophosphorylated RNA (see section 2.8.1, above) was incubated in RNase Assay Buffer (section 2.3) at 50°C for 2 min, followed by a 10 min incubation at 37°C. RNA was chilled on ice, followed by incubation at 30°C for 30 min with 2.4 pmoles purified ribosomal proteins S20 (gift from G. A. Mackie) and S4 (gift from Drs. Kellie Horn and David Draper) as indicated in the Figure Legends. Reactions were initiated by addition of 18 pmoles purified RNase G and allowed to proceed as outlined in section 2.8.3. 2.8.5. Competition assays Reactions were performed as described in Section 2.8.3, but unlabelled oligonucleotide competitor (quantities listed in Figure Legends) was added concurrently with enzyme. Competitors were either used in their unphosphorylated condition (5'-OH group) or the oligonucleotides were 5'-monophosphorylated with cold ATP, as described in Section 2.8.2. Competitor sequences are listed in Fig. 5.2. 58 2.8.6. Phosphatase assays and thin-layer chromatography 5 uCi Y 3 2 P - A T P , 8 pmol A T P were incubated at 50°C for 2 min, 37°C for 10 min in RNase Assay Buffer (Section 2.3). The reaction was initiated by the addition of 400 ng pnp13 degradosome in a final volume of 30 ul. At 0, 30 and 90 min, 8 ul aliquots were removed and heated at 100°C for 2 min. To examine phosphatase activity on labelled 5'-monophosphorylated oligonucleotides, assay reactions were performed as described in Section 2.8.3, but at each time point, 6 pi of the assay volume was removed and heated at 100°C for 2 min. As a positive control, 8 pmol ATP containing 5 pCi [y32P]-ATP was incubated at 37°C for 45 min with 0.1 U/pl calf intestinal phosphatase (Pharmacia) in Buffer III (Section 2.3) in a final reaction volume of 10 pi. Heating at 75°C for 10 mins inactivated calf intestinal phosphatase. Polyethyleneimine-impregnated 5x10 cm thin layer chromatography plates (Macherey-Nagel) were rinsed in dH20, and allowed to dry. 1-1.5 pi of each sample was spotted on to the plate approximately 1 cm from the bottom of the plate. The plates were placed in a chromatography chamber containing approximately 0.5 cm 0.375 M KH 2P0 4 (pH adjusted to 3.5 with HCI) and the solvent run until within 2 cm of the top of the plate. Plates were dried, and products visualized by phosphorimaging (Molecular Dynamics). 59 3. ACTIVITY AND QUATERNARY STRUCTURE OF RNase G 3.1. Introduction RNase G is the endonuclease responsible for formation of the mature 5'-end of 16S rRNA from a larger precursor (Li et al., 1999b; Wachi et al., 1999). In addition, RNase G may control the stability of alcohol dehydrogenase and enolase mRNAs (Kaga et al., 2002; Umitsuki er al., 2001). Moreover, array data suggest that RNase G can also act on a limited set of additional mRNAs (Lee et al., 2002). Curiously, the gene encoding RNase G was originally identified as Caf A which when overexpressed resulted in the formation of cytoplasmic axial filaments in E. coli (Okada et al., 1994). Importantly, RNase G exhibits 35% sequence identity and 50% similarity to the catalytic domain of RNase E (McDowall and Cohen, 1996; McDowall etai., 1993; Taraseviciene etai., 1995). RNase G can mimic some RNase E cleavages in vitro, and these cleavages are also 5'-end-dependent (Jiang et al., 2000; Tock et al., 2000), a characteristic of RNase E activity (Mackie, 1998). RNase E is the major endonuclease in E. coli and has been implicated in rRNA processing (Li era/., 1999b; Misra and Apirion, 1979; Wachi etai., 1999), tRNA maturation (Li and Deutscher, 2002; Ow and Kushner, 2002) and bulk mRNA decay (see reviews (Coburn and Mackie, 1999; Grunberg-Manago, 1999; Regnier and Arraiano, 2000; Steege, 2000)). RNase E associates in vivo with PNPase, a 3' to 5'-exonuclease, Rhl B, a DEAD-box RNA helicase, and enolase, a glycolytic enzyme, to form the RNA degradosome (Carpousis et al., 1994; Py et al., 1996). The assembly of the degradosome requires the C-terminal domain of RNase E (residues 645 to 1045) to serve as a scaffold 60 (Vanzo et al., 1998). However, this domain has no equivalent in RNase G and is not required for endonucleolytic activity. Several important questions still surround RNase E and RNase G. First, the active site in either enzyme has yet to be identified. Second, the basis of substrate recognition by both enzymes remains elusive. Both cleave RNA at single-stranded sites rich in A and U residues (Ehretsmann et al., 1992; Jiang et al., 2000; Mackie, 1991; Mackie, 1992; McDowall etai., 1994; Tock etai., 2000). RNase G, however, appears to cleave somewhat more promiscuously within a given sequence than does RNase E (Tock et al., 2000). The molecular basis for these findings is unknown. Finally, the tertiary and quaternary structures of both enzymes are unknown. Potential self-interactions within RNase E have been demonstrated with yeast two-hybrid experiments (Fig. 1.4; Vanzo et al., 1998), implying that RNase E may form homodimers, but this has not been confirmed by other means. RNase G shares significant similarity to the N-terminal domain of RNase E, the region associated with endonucleolytic activity and self-interaction (McDowall and Cohen, 1996; McDowall etai., 1993; Taraseviciene etai., 1995; Vanzo et al., 1998). This implies that the properties of RNase G will likely also pertain to the catalytic domain of RNase E, allowing RNase G to act as a surrogate for investigating the structure and activity of RNase E. 61 3.2. Results 3.2.1. Sequence comparison A number of regions of high similarity (90% or greater) are visible in the alignment (Fig 3.1) of RNase G and RNase E from a number of different species. Regions of interest are described in Table 3.1. The first region of high similarity is located between residues 100 and 110 (with respect to the residues of RNase G from E. coli). This is located in the S1 domain, which extends from approximately residue 35 to 135. This is a potential RNA binding domain, which was originally identified in ribosomal protein S1 (reviewed in Coburn and Mackie, 1999). Two temperature-sensitive alleles (marked with \"t\") of RNase E also map to this region, ams-1 (G66S) and rne-3071 (L68F)(McDowall et al., 1993). Residue 267 is marked with an arrow, corresponding to the truncated RNase G protein which is produced by the BUMMER strain (Wachi et al., 1999). The second region of high similarity is located between residues 279 and 351, and includes the highly conserved aspartic acids at residues 304, 347 and 350, which are marked with \"#\". Recent findings in our lab have revealed that introducing D304A or D347A mutations lead to a loss of activity, while a D350A mutation leads to a decrease in activity (V. Kunanithy and G. A. Mackie, unpublished). The third region of high similarity spans residues 377 to 400. The cysteine residues are indicated with \"@\" symbols, and it is interesting to note that apart from C405 and C408, which are highly conserved, there is very little conservation among cysteine residues. 62 Figure 3.1. Sequence alignment of RNase G and RNase E. Organism names are listed to the left of each sequence, and the sequences are grouped into RNase G and RNase E homologues. Sequences were aligned using ClustalW. Numbers at the top correspond to amino acid residues in E. coli RNase G, and numbers to the right of each sequence indicate the actual residue number for each sequence. Residues highlighted in black are similar in over 90% of the proteins, dark grey residues are similar in 75-90% of proteins and light grey residues represent 50-75% similarity. The six cysteine residues found in E. coli RNase G are marked with \"@\" above the sequence. Aspartic acid residues which have been mutated to alanines by this lab (V. Kunanithy and G. A. Mackie, unpublished) are marked with \"#\". Two temperature sensitive mutations, ams-1 (G66S) and rne-3071 (L68F) are market by \"t\". The three high similarity regions (HSR1-3) are marked with horizontal arrows, as is the S1 domain. A vertical arrow marks the position of the C-terminus of the truncated RNase G encoded by the BUMMER strain. 63 S1 halodurans C. trachomatis H. influenzae W. meningitidis P. aeruginosa V. cholerae X. fastidiosa A. aeolicus N. gonorrhoeae A. actinomycetem* E. coli R . .ipii t [j i , \\ ' : .1 H. influenzae M. tuberculosis N. meningitidis P. aeruginosa F. prowazekil Synechocystis sp V. cholerae X. fastidiosa A. thaliana G . the ta N. [REARRGI' ; R P V E E R I V f S | j f ePKKI RQLKGW| JTHEP.KKVP. Q LKGN| g l j J E B K K I P Q L K r a | LRQAKPGIVG!l | NSEHSLVSMg [ P T Q R P G I v q ^ LEAKRGI' ;RDGCP.( FK^ERRKEKYPTGAF: 8 0 •QEG SJ^tPHTECVAGEEQKQFTVRDj S F H L S N E E E D E K K - - K R N | | S 0 F 1 ( ENSKKFEQMFDIDTSEAr?; ; KELgK:. . ; ENSKKFEQMFDMDSEEAP|EEF»I|LD E N S K K F E Q M F D I D T S K A P | | E E L B K L D fVSHTECVDENEQKQFKVKsffsEIflREG EQPRNPEE jSN REGSAVES S|SA£lHE I|«5TALH|Lgl|QEEAMKDNTGEVRAQVPVD®»T FtLl*EKRAL FAME ERLD - V N S V L I P N I H L E N P H J & I N R O iVHfliS L A E i L B E E E A L K D RTAEVRARVP F(3glAFliNEKRNITKIELRTR-AJ^Fn.PDDHLETPH&VQR5 ^SANAMLJ^iT|ENEIFEERIDin^TNIVSVIYLIJ4NKRAIKFIEEKY^ .LPGEKGFgpLSPTAWSSIKVNDlPKKEEAKfeSP-tDLLFHNYQEOGDRDSNRRRRRRRGSEFSEKENXKSg iNRSLAL SALSLgEEEAL K D N T S CVLAVVPVSgftS YIiNEKRRINHIEKAQQ-VR?fTXVPNSDMETPH^VIRa l I p L S L S | l | l | E E H A M K E N T G Q V L V Q T P V E ^ Y L L ^ K R R I N E I E K R H D - A P f i I I I A D E O | E A I ^ T F S K | E C E | C R Q L V S V IIsrj$FCYFSFQPNEEHlJ I S K S S F L Y D R | L S L N K N L F K T N LIIKYTLFSNVKLIYNY -DSNYFHFFIQL EQLDSIKP5SYKS KRisSSVKS^FSE-lDMINRFFKK-- - L S N F § Y A R N ^ 489 R N a s e G - i s : 4 7 8 4 7 4 4 8 0 4 7 9 I B' B ' ' ' \" H ' ; 'fl™L||QP3SAGGGGGKRRKRARETEVAAEvAEPVALPAKAEAAPAAPTAQDVTVREE|ERPV^ ; t F T R § ? n j J s ^ T ; | K f L S . Q K I L f K | g I R E D L K K L K D F E E V r I i K V H P N ' | S G Y F K R E D I K K L Q K E F K | K L N L D Y G W H D P N S Y E I K A : 65 Table 3.1. Features of RNase G and RNase E. Several features were noted when comparing the amino acid sequence alignment of RNase G and RNase E from a variety of prokaryotic organisms (Fig. 3.1). The amino acid residue numbers are based on the sequence of RNase G from E. coli. HSR is used to describe High Similarity Regions. Feature residue number(s) importance S1 domain 35-135 HSR1 100-110 HSR2 279-351 HSR3 377-400 ams-1 66 rne-3071 68 BUMMER 267 cysteine 79, 162, 402, 405, 408, residues 421 aspartate 304, 347, 350 residues potential RNA-binding domain, possible role in 5'-end dependence unknown possible role in endonuclease activity unknown Gly—>Ser results in temperature sensitivity in RNase E Leu-»Phe leads to temperature sensitivity in RNase E truncation at this residue results in a loss of activity mutant of RNase G unknown function, useful as a tool for elucidating quaternary structure 304 and 347 required for activity of RNase G, 350 affects activity. Possible metal binding residues 66 3.2.2. Purification of RNase G and identification of its translational start site. Purification of His-tagged RNase G has been described previously (Jiang et al., 2000; Tock et al., 2000). Neither report, however, resolved the ambiguity of the two potential sites for translation initiation that reside 18 nucleotides apart (Li et al., 1999b; Wachi etal., 1999) in the rng gene, as shown in Fig. 3.2. The cloning strategies used by others have utilized artificial upstream regions, thereby selecting for a particular start site (Lee et al., 2002; Tock et al., 2000). Furthermore, the purification of RNase G by Tock et al. (2000) was performed under denaturing conditions. Two separate cloning and purification strategies were undertaken to determine the correct N-terminus of RNase G. To examine full length, untagged RNase G, the PCR strategy outlined in Fig. 3.2 (also see Materials and Methods, section 2.6.1) was used to maintain the integrity of both potential N-termini of RNase G. By retaining 64 nucleotides upstream of the second potential start site in the cloned fragment, the bacterial host was allowed to select the preferred N-terminus. The rng gene was amplified by PCR from genomic DNA from strain CF881 (Table 2.1) using the primers listed in Table 2.2 which add BamHI sites to the 5'- and 3'- ends. The amplified DNA was cut with BamHI and ligated into the corresponding site of pET11. Following transformation and isolation of clones, the correct orientation of the insert was confirmed by restriction endonuclease mapping and the resulting plasmid was 67 amplified fragment > -18 +1 +1469 -64 51 UAA +1507 rng gene M T A E L L V N... 55364 Da M R K G I N M T... 56064 Da Figure 3.2. Potential translation initiation sites of the rng gene. The rng open reading frame is represented by an open box together with two possible translation initiation sites shown by horizontal arrows. Nucleotides are numbered beginning with the first residue of the open reading frame determined in our experiments (+1, see Results 3.2.2). The site at +1 corresponds to an AUG start codon, with the N-terminal sequence and predicted molecular weight listed below the open reading frame. The second potential site of initiation is a GUG codon which begins 18 nts 5' to the +1 site and would generate the N-terminal sequence and molecular weight indicated at the bottom of the diagram. The region of the rng gene amplified by PCR (see Section 2.6.1) encompasses 64 nts 5' to the +1 site, and 23 nts 3' of the translational stop codon. 68 designated pDB6. In an attempt to preserve both the structure and activity of wild type, untagged RNase G, denaturation steps were avoided during the purification. The purification is described in section 2.6.1. Fractions from successive steps of the purification are shown in Fig. 3.3, with each lane representing roughly equivalent amounts of protein. The initial step in the purification was a 30,000 x g centrifugation to remove insoluble proteins and cellular debris. The insoluble fraction (Fraction 1) contained a significant portion of the overexpressed RNase G, presumably due to misfolding or association of the protein with the cellular membrane (Fig. 3.3, lane 2). In this regard, the N-terminal region of RNase E, which shares a high similarity to RNase G, also interacts with the membrane (Liou et al., 2001). This fraction of the protein may also represent RNase G associated with cytoplasmic axial filaments (cited in Tock et al., 2000). RNase G was the most abundant protein in the soluble S30 fraction (Fraction 2), although there were a number of contaminants (Fig. 3.3, lane 3). RNase G in Fraction 2 was precipitated with 26% w/v ammonium sulfate (approximately 40% of saturation) leading to the removal of several visible contaminants, including a prominent 31 kDa band (Fig. 3.3; compare lanes 3 and 4). Further impurities were largely removed by affinity chromatography on a Heparin-agarose column, and the resulting fractions were pooled to maximize purity, rather than yield, forming Fraction 3 (Fig. 3.3, lane 5). The final step in purification was anion exchange chromatography (see Materials and Methods, section 2.6.1). The resulting fractions were also pooled to maximize purity yielding Fraction 4 (Fig. 3.3, lane 6). This step removed most of the remaining 69 kDa 200-116-97.4-66 - \\ 1 2 3 4 5 6 45 H 3 H 'RNase G Figure 3.3. Purification of RNase G. Purification of untagged RNase G is described in Materials and Methods, section 2.6.1. Each lane contains approximately 500 ng of total protein. Lane 1 contains size markers, with sizes indicated to the left of the panel. The remaining lanes are as follows: lane 2, protein from the 30,000 x g pellet (Fraction 1); lane 3, Fraction 2, 30,000 x g supernatant; lane 4, Fraction 3, AS-26; lane 5, Fraction 4, heparin column; lane 6, Fraction 5, final pooled fractions from anion exchange chromatography on a Source Q (Pharmacia) column. Proteins were separated on a 10% SDS-polyacrylamide gel and visualized by staining with Coomassie Brilliant Blue R-250. 70 impurities. Yields of up to 6 mg/l culture were obtained by this method. The gain in specific activity during purification could not be determined reliably. Representative elution profiles for the heparin and anion exchange columns are shown in Appendix Figure 1, panels a and b, respectively. The second cloning strategy was used to overexpress and ultimately purify RNase G with a C-terminal 6xHis tag. This strategy is depicted in Fig. 3.4. To construct the overexpression vector, pET24b (Novagen) was isolated from the dam' strain JM110 and digested with Xbal (dam methylation-sensitive) and Nhel and religated to remove the ribosome binding site, thus creating pET24b-mod. The rng gene was amplified by PCR from genomic DNA from E. coli strain CF881 (Table 2.1) using the primers listed in Table 2.2. This introduced BamHI and Xhol sites into the 5'- and 3'- ends, respectively. The resulting fragment was cleaved with Xhol and BamHI and ligated between the corresponding sites of pET24b-mod producing the recombinant plasmid pDB1. As a consequence of these manipulations, the RNase G enzyme encoded by pDB1 contains a non-cleavable His6 tag at its C-terminus. His-tagged RNase G was purified under non-denaturing conditions, as described in section 2.6.2. As shown in Fig. 3.2, translation of RNase G could be initiated at either of two in-frame sites. The potential N-terminal sequences are MRKGINM and MTAELLV resulting from initiation at the -18 or +1 sites in the rng gene, respectively. Two lines of evidence show that only the second site is authentic. First, purified RNase G was subjected to electrophoresis and blotting to a PVDF membrane (see section 2.7.3). The region containing RNase G was excised and gas phase sequencing was performed (University of Victoria-Genome B. C. 71 Figure 3.4. Construction of pDB1. Plasmid pDB1 was constructed as described in the text, section 2.6.3. Panel a shows the intact plasmid pET24b. The region of interest is expanded in panel b, with the T7 RNA polymerase promoter indicated with an arrow facing the direction of transcription, the ribosome binding site indicated as a box with diagonal stripes, and the region encoding the six histidine tag as a shaded box. Religation of the plasmid cut with Xbal and Nhel results in plasmid pET24b-mod, which is shown in panel c. The \"X\" indicates the loss of the Xbal and Nhel restriction endonuclease sites at the ligated junction of the plasmid. Panel d shows the plasmid pDB1 with the rng coding sequence inserted between the BamHI and Xhol sites. Expression adds six histidines to the C-terminus of RNase G. 72 cut with Xbal/Nhel religate T7 promoter X _l_ BamHI I cut with BamHI/Xhol add insert ligate Xhol His-tag BamHI Xhol rng promoter X BamHI J Xhol rng Hs-tag 73 Proteomics Centre). The sequence obtained, TAELLV, corresponds to translational initiation at the second of two potential start sites (+1) in the rng gene followed by removal of the N-terminal methionine by methionyl amino peptidase (Ben-Bassat et al., 1987). Interestingly, earlier attempts to characterize Caf A/RNase G resulted in purification of a 51 kDa protein whose N-terminus was TAELLVNVTP (Okada et al., 1994). Second, the expected mass of RNase G translated from the -18 site is 56,064 while it would be 55,364 if translated from the +1 site. If the N-terminal methionine is cleaved from the protein, the expected mass in the second case would be 55,233. The mass determined by electrospray mass spectrometry (Table 3.2, section 2.7.4) was 55,228, which corresponds to initiation at +1 followed by cleavage of the N-terminal methionine. No species of mass corresponding to initiation at the potential translational start site at the -18 position were detected. 3.2.3. Oligomerization of RNase G The quaternary structure of RNase G was probed because the resultant information would also provide insight into the structure of RNase E. Preliminary results showed that RNase E could be cross-linked by mild oxidation (J. S. Hankins and G. A. Mackie, unpublished data), which indicates that cysteines are located in close proximity between subunits at the protein-protein interface. We employed Cu2+-phenanthroline to introduce inter-molecular cross-links into His-tagged RNase G and resolved the products by two-dimensional electrophoresis (section 2.7.5). The resultant partially oxidized species were separated in the first dimension under non-reducing conditions (Fig. 3.5a, b). The prominent band 74 Table 3.2. Mass spectrometer analysis and sequencing of RNase G mutants. Mass spectrophotometry was performed as described in Materials and Methods, section 2.7.4. The expected mass corresponds to the mass calculated, using ProtParam (http://us.expasy.org/tools/protparam.html), for each RNase G mutant with the N-terminal methionine (fMet) cleaved. Potential RNase G cysteine to serine mutants were sequenced through the region of interest to confirm success of the site-directed mutagenesis (Materials and Methods, sections 2.4, 2.6.2). expected determined mass mass confirmed by ENZYME (-fMet) (+/- 0.02%) difference sequencing wt 55,233 55,228 +5 yes wt, His-tagged 56,298 56,312 +14 yes C405S/C408S 56,265 56,273 +8 yes C421S 56,282 nd nd yes C402S/C405S/C408S 56,250 56,266 +16 yes C402S 56,282 56,304 +22 yes C162S 56,282 nd nd yes AS1 45,401 45,415 +14 yes 75 1 w \\ 1 1 1 1 1 [* 200- M 1 116- mm 97.4- *•) 66- H * 45-dimer monomer reduced monomer d imer Figure 3.5. Oxidative cross-linking of RNase G. Samples (3 pg) of His-tagged RNase G were oxidatively cross-linked by C u 2 + -phenanthroline as described in Materials and Methods, section 2.7.5. Panels a and b depict 2-dimensional analysis of 3 pg of each cross-linked His-tagged RNase G. In both panels, the first dimension is non-reducing. Monomeric RNase G is denoted by *. The second dimension gel in Panel a is non-reducing, and in Panel B the second dimension gel contains 100 mM DTT, and is thus reducing. Proteins in Panels a and b were visualized by staining with Coomassie Brilliant Blue R250, and monomeric and dimeric species are indicated by arrows. These experiments were performed by Janet S. Hankins and were published in Briant et al. (2003). 76 at 56 kDa (denoted by *) represents monomeric RNase G. The higher molecular mass bands represent intermolecularly cross-linked RNase G, including a distinct band migrating at the position corresponding to RNase G dimers. A number of higher multimers, some the size of tetramers, were also observed in Fig. 3.5a. These results suggested that RNase G exists as at least a dimer. In addition to these more slowly migrating species, a second, faster running band was observed just ahead of the monomer (Fig. 3.5a, b). This apparently resulted from the formation of intramolecular disulphide bonds (see below). In Fig. 3.5a, the second dimension polyacrylamide gel was non-reducing and all detectable species separated along a diagonal, consistent with maintenance of disulphide bonds. Due to incomplete stacking, the samples were streaked. In Fig. 3.5b, cross-linked samples were initially separated under non-reducing conditions, but reduced in DTT prior to application to the second dimension. On the second dimension gel, all species from the first dimension, including that migrating faster than the monomer, migrated with an apparent mass of 56 kDa, rather than on a diagonal. This result demonstrates the reversibility of the oxidative cross-link and clearly shows that the mobilities of the various species in Fig. 3.5a must result from differences in disulphide bonding. Sucrose gradient sedimentation velocity centrifugation was also employed to determine the multimeric state of wild-type RNase G (see Materials and Methods, section 2.7.7). Fig. 3.6 shows the sedimentation profile of wild-type RNase G relative to markers in a 5-20% w/v sucrose gradient in the presence of DTT. The bulk of RNase G sedimented at a velocity which was consistent with a mass of around 130 kDa, which corresponds to a dimer. Static 77 158kDa 67kDa aldolase BSA 1.2 T j 1— 9 10 11 12 13 14 15 16 17 18 19 20 fraction Figure 3.6. Sedimentation velocity analysis of RNase G. 30 ug of purified RNase G (untagged; •) was analyzed by sedimentation through a 2 ml 5-20% w/v sucrose gradient as described in Materials and Methods, section 2.7.7. Fraction numbers are indicated at the bottom of the panel. The elution profiles of 4 pg each of aldolase (158 kDa, • ) and bovine serum albumin (BSA, 67 kDa, •) are represented as dotted lines. The peak fraction for each standard is indicated with a vertical line. Sedimentation is from right to left. 78 light scattering of RNase G also demonstrated that RNase G was multimeric, but heterogeneity of the sample prevented an accurate determination of its size (data not shown). An estimate of the size of RNase G was also made by gel filtration (Materials and Methods, section 2.7.8; Appendix Fig. 2). The majority of RNase G eluted from a calibrated Superdex 200 column (Pharmacia) at an elution volume corresponding to approximately 210 kDa (Fig. 3.7). This indicated that RNase G was likely tetrameric under the experimental conditions. The double peak following elution of each sample corresponds to components of the buffer (Appendix Fig. 2). 3.2.4. Effect of cysteine to serine mutations in RNase G RNase G and RNase E from different organisms share a number of conserved residues. Strikingly, those corresponding to cysteine residues 405 and 408 of E. coli RNase G are conserved in 26 of the 29 RNase E and RNase G protein sequences aligned (see Figure 3.1, and section 3.2.1). Moreover, the oxidative cross-linking of RNase G shows that cysteines on different subunits must be in proximity. RNase G from E. coli contains six cysteine residues. Figure 3.8 is a schematic diagram of RNase E and RNase G, with the relative positions of their cysteine residues indicated. Cysteines at position 405 and 408 (relative to RNase G) are highly conserved. RNase G contains one cysteine (C79) in the S1 domain. Each cysteine was systematically changed to serine alone or in combination (see Materials and Methods, section 2.6.2), creating the mutants listed in Table 2.2. This was done to determine which cysteines were responsible for crosslinking, and thus which cysteines lie in close proximity. 79 Figure 3.7. Gel filtration analysis of RNase G. 640 ug wt RNase G, 960 ug RNase G C402S/C405S/C408S, 150 ug ug RNase G-His6 and 70 ug RNase G C405S/C408S were examined by gel filtration in 25 mM Tris-HCl pH 7.6, 320 mM NaCl on a calibrated Superdex S200 column (Pharmacia) as described in Materials and Methods, section 2.7.8. The standards, marked by •, were thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), BSA (67 kDa), ovalbumin (45 kDa) and chymotrypsin (25 kDa) (all standards from Pharmacia). Sizes of the RNase G mutants were estimated from their elution volume (Appendix Fig. 2), and are marked with dotted lines. Wild-type RNase G eluted at 11 mis, RNase G C405S/C408S at 12.7 mis, RNase G-His6 at 12.9 mis and RNase G C402S/C405S/C408S at 13.4 mis. The column was calibrated by R. Pfeutzner. 80 Fig 3.7 Figure 3.8. Structural comparison of RNase E and RNase G, and the locations of cysteine residues. A schematic of RNase E is shown at the top of the figure, and RNase G at the bottom. The C-terminal scaffolding domain of RNase E is shaded, and the S1 domains of both RNase E and RNase G are indicated by diagonal striping. The numbering above each diagram corresponds to the amino acid residue. All cysteines are identified with stars. In RNase E, cysteines are found at residues 404, 407, 471 and 832. In RNase G, cysteines are located at residues 79, 162, 402, 405, 408 and 421. For complete sequences of RNase G and the N-terminus of RNase E, see Fig. 3.1. 82 83 3.2.4.1. Physical properties of RNase G cysteine to serine mutations Each mutant protein was expressed and tested for solubility following centrifugation at 30,000 x g. All RNase G mutants listed in Table 2.2 produced soluble proteins. In contrast, RNase G C79S could not be overexpressed while RNase G C402S/C405S/C408S/C421S and RNase G C405S/C408S/C421S were insoluble or marginally soluble. Introduction of the desired mutations was confirmed by sequencing through the region of interest; several purified RNase G mutants were also analyzed by mass spectrometry (Table 3.2). Oddly, the mass of each His-tagged RNase G determined by mass spectrometry is higher than the expected mass by approximately 14 Da, the mass of a methyl group. Presumably a mutation was been introduced into the parent strain, pDB1, during the PCR-based site-directed mutagenesis reaction (Materials and Methods, sections 2.4 and 2.6.2). The parent plasmid (pDB1) was completely sequenced through the rng coding region, and no mutations were observed. The increased mass could, therefore, be due to a mutation in the C-terminal tag, which was then carried over to the subsequent plasmids encoding RNase G C405S/C408S, C402S and C402S/C405S/C408S. Samples of each soluble mutant were separated on an SDS-polyacrylamide gel (Fig. 3.9). While each preparation was close to homogeneous, each contained traces of contaminating proteins. The increased mobility of wild type RNase G, Fig. 3.9, lanes 14, 15, was due to its smaller size, since it lacked a His6 tag. Western analysis (Fig. 3.10) was used to illustrate that each mutant was recognized by an anti-RNase G polyclonal antibody, while the negative controls, catalase (Fig 3.10, lane 1) and aldolase (Fig 3.10, lane 2) did 84 O' O 0 © O O & 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Figure 3.9. Purity of RNase G mutants. Duplicate samples of each RNase G mutant preparation (section 2.6.2) were separated on a 10% SDS-polyacrylamide gel, and stained with SYPRO red (Pharmacia) for quantitation. The position of RNase G is indicated to the right of the gel with an arrow. Lane 1 contains standards; lanes 2 and 3, 100 ng RNase G-His6 (section 2.6.3); lanes 4 and 5, 260 ng RNase G C405S/C408S; lanes 6 and 7, 210 ng RNase G C421S; lanes 8 and 9, 200 ng C402S/C405S/C408S; lanes 10 and 11, RNase G C402S; lanes 12 and 13, RNase G C162S; lanes 14 and 15, wild type RNase G (section 2.6.3). 85 ^56 kDa 1 2 3 4 5 6 7 8 Figure 3.10. Western blotting of RNase G variants with cysteine to serine mutations. 64 ng of each protein sample were separated on a 10% SDS-polyacrylamide gel, blotted to nitrocellulose and probed with anti-RNase G antibodies as described in Materials and Methods, section 2.7.2. Lanes 1 and 2 contain the negative controls catalase and aldolase (Pharmacia). Lane 3, RNase G C405S/C408S; lane 4, C402S/C405S/C408S; lane 5, wild-type RNase G; lane 6, His-tagged RNase G; lane 7, RNase G C421S; lane 8, RNase G C402S. The position of migration of RNase G is indicated to the right by an arrow. 86 not bind the antibody. RNase G C421S (Fig. 3.10, lane 7) was either under-loaded or poorly recognized by the antibody. To ensure that no gross significant structural changes had been introduced into the mutant RNase G proteins, the midpoint unfolding temperature (Tm) of each RNase G variant was determined by circular dichroism (see Materials and Methods, section 2.7.6). The Tms for each mutant are compiled in Table 3.3. Unfolding was irreversible. All His-tagged RNase G proteins examined displayed significantly lower Tm values compared to wild-type RNase G (Tm=54.8°). RNase G-His6 and RNase G C405S/C408S exhibited Tm values just above 50°. In contrast, RNase G C402S and RNase G C402S/C405S/C408S showed Tm values just below 49°, while RNase G C402S and RNase G C162S had a Tm below 47°. This lowering of the Tm may reflect either a decrease in the stability of the enzyme, or a change in the multimeric state of the protein. Therefore, it can be inferred that addition of the His6 tag reduced the stability of RNase G, and that each of the examined cysteine to serine mutations also led to a modest decrease in structural stability, or these changes affected the multimeric state. Each RNase G mutant was also subjected to oxidative cross-linking and separated on a one-dimensional, non-reducing polyacrylamide gel. Representative samples are shown in Fig. 3.11 and lane 2 contains a sample of RNase G-His6 for comparison. The pattern of cross-linking was equivalent to that observed in the first dimension of Fig. 3.5a. A cross-linked sample subsequently treated with 0.05 M DTT migrated as a sharp, homogeneous band 87 Table 3.3. Comparison of RNase G mutants. Tm were determined by circular dichroism and activity was determined as described in Experimental Procedures. The 671 nt and 728 nt pre-rRNA substrates were transcribed from pGM122 and pGM119 linearized with BamHI, respectively (Figure 2.2e, d). ENZYME Tm (°C)a Relative Activity b 671 nt 728 nt 9S wt no tag 54.8 +/- 0.2 100 100 100 wt, His-tagged 50.3+/-0.1 110 190 60 C405S/C408S 50.1 +/- 0.1 160 190 55 C421S 45.1 +/-0.3 40 45 25 C402S/C405S/C408S 48.6+/-0.1 20 45 25 C402S 48.7 +/-0.1 50 60 60 C162S 46.8 +/- 1.0 35 45 25 a Tm values are the average of duplicate experiments b Relative Activities are the average of triplicate experiments 88 1 2 3 4 5 6 7 8 Figure 3.11. Crosslinking of RNase G mutants. A one-dimensional separation of cross-linked mutant RNase G proteins was performed on a non-reducing SDS-polyacrylamide gel stained with SYPRO Red (Pharmacia). Lane 1 contains size markers; lanes 2-7 contain, respectively: RNase G-His6; RNase G C405S /C408S; RNase G C 4 2 1 S ; RNase G C402S /C405S /C408S; RNase G C 4 0 2 S and RNase G C 1 6 2 S . Lane 3 contains approximately 6 pg protein, while lanes 2 and 4 - 7 contain 3 ug. The size of monomeric RNase G (56 kDa) is indicated at left, and the two intramolecular species, bands i and ii, are identified to the right of the panel. The experiment was performed by Janet S. Hankins, and is described in Materials and Methods, section 2.7.5. 89 with an apparent size of 56 kDa (data not shown). Thus, the band migrating slightly faster than the 116 kDa marker in Fig. 3.11, lane 2 would represent apparent dimers. Bands i and ii, migrating slightly faster than monomers (56 kDa), represent intramolecularly cross linked RNase G. RNase G C405S/C408S (Fig. 3.11, lane 3) also formed at least two different intramolecular cross-linked bands. Both migrated to the same position as bands i and ii in Fig. 3.11, lane 2. A series of multimers was also formed by oxidation of RNase G C405S/C408S, notably, apparent dimers that migrated slightly faster than the 116 kDa marker and apparent tetramers (slightly slower than the 200 kDa marker; Fig. 3.11, lane 3). Intermediate bands between dimers and tetramers likely resulted from a mixture of inter- and intramolecular bridged species. The distribution and relative intensities of cross-linked species formed from RNase G C402S did not differ from those in wild-type RNase G and RNase G C405S/C408S (Fig. 3.11; compare lane 6 with lanes 2 and 3). Likewise, RNase G C402S/C405S/C408S, which combines the previous mutations, retained the ability to undergo oxidative dimerization as evidenced by a faint band just below the 116 kDa marker (Fig. 3.11, lane 5). However, bands i and ii migrating ahead of the 56 kDa monomeric band could not be detected. Oxidative cross-linking of RNase G C162S revealed apparent dimers and band ii (Fig. 3.11, lane 7). Taken together, these data imply that upon oxidation different cysteine residues within a given RNase G monomer are capable of forming intermolecular and intramolecular disulphide bonds. Useful information was not obtained for RNase G C421S as it was poorly soluble in the buffer used for cross-linking (Fig. 3.11, lane 4). An interpretation of these data is provided in the Discussion (section 3.3). 90 Figure 3.12. Sedimentation velocity analysis of RNase G mutants. 30 ug of purified RNase G (untagged; •), RNase G-His6 (•), RNase G C405S/C408S (A) or RNase G C421S (•) were analyzed by sedimentation through a 2 ml 5-20% w/v sucrose gradient as described in Materials and Methods (section 2.7.6). Panel a depicts the elution profile. Fraction numbers are indicated at the bottom of the panel, and the position of sedimentation of the standards aldolase (158 kDa) and BSA (67 kDa) are indicated with vertical lines. Sedimentation is from right to left. In Panel b, the activity was tested for each mutant. For each sample, 2 pi of wt RNase G (Fraction 14), RNase G-His (Fraction 15), RNase G C405S/C408S (Fraction 15) or RNase G C402S/C405S/C408S were incubated with 60 fmoles labelled monophosphorylated 519 nt RNA (Fig. 2.1) for 4 hrs, as described in Materials and Methods, section 2.8.3. Products of the assay were separated on an 8% polyacrylamide gel. Lanes 1 and 2 are negative controls, showing the substrate at 0 hrs and 4 hrs (T=0 and T=4, respectively). Lane 3 is a sample from the RNase G-His6 assay, lane 4 from the RNase G C405S/C408S assay, lane 5 from the RNase G C402S/C405S/C408S assay and lane 6 from the wt RNase G assay. Intact substrate is indicated by \"S\", and the major products of endoribonucleolytic cleavage are marked with arrows to the right of the gel (P). 91 92 The sedimentation rates of RNase G, RNase G-His6, RNase G C405S/C408S and RNase G C402S/C405S/C408S were compared to provide an independent measure of their sizes (Fig. 3.12a). RNase G-His6 and RNase G C405S/C408S both sedimented at a rate that would correspond to dimers. RNase G C402S/C405S/C408S sedimented just ahead of BSA, indicating that it forms monomers. These RNase G mutants were also sized by gel filtration chromatography. In Fig. 3.7, RNase G-His6 was estimated to be 105 kDa, and RNase G C405S/C408S as 110 kDa, which both correspond to dimers. In contrast, RNase G C402S/C405S/C408S was estimated to be 85 kDa from Fig. 3.7, which confirmed that this mutant behaved as a monomer. It is important to note that both sedimentation velocity centrifugation and gel filtration separate proteins based on both size and shape. The samples may also undergo some auto-oxidation during the course of the experiment. Sizes of complexes are, therefore, estimates. 3.2.4.2. Activity of wild type RNase G and mutants The ability of degradosomes, wild type RNase G or RNase G C405S/C408S to cleave two model substrates, 9S RNA and rpsT mRNA, was determined (Fig 3.13). Assays using 9S RNA as substrate are shown in Panel c. Fig. 3.13c lanes 5 and 6 contain degradsosomes, and are a positive control. Bands corresponding to products of cleavage at the \"a\" site accumulate, along with a fainter band containing pre-5S rRNA, which is the final product of RNase E processing. Despite their strong sequence conservation, mutation of C405 and C408 actually increased activity on these substrates RNase G-His6 (compare 93 Figure 3.13. Comparison of rpsT mRNA and 9S processing sites. Panel a is a schematic of 9S RNA. 9S RNA undergoes RNase E-mediated cleavage at sites a and b to release pre-5S RNA (stem-loop III). RNase E makes three major cleavages in the rpsT mRNA, and these are indicated in Panel b as sites a-c (minor cleavage sites are not shown). Panels c and d shows the products of endonuclease assay (section 2.8.3) of 0.4 pmoles 9S and rpsT mRNA, respectively, following separation on an 8% urea-polyacrylamide sequencing gel. In panels c and d, lane 1 is the 0 min control. Lane 2 contains products following treatment with 14 pmol RNase G-His6; lanes 3 and 4, 14 pmol RNase G C405S/C408S. Lanes 5 and 6, containing 100 ng wild-type degradosome serve as positive controls. Panel c shows the processing of 9S RNA, and panel d shows the products of cleavage of rpsT mRNA. 94 95 Fig 3.13c, compare lanes 2 and 4). The products resulting from cleavage at the \"a\" site and pre-5S rRNA are both visible over the course of the assay. Interestingly, significant amounts of pre-5S rRNA product did not accumulate over the course of the assay with ether RNase G-His6 or RNase G C405S/C408S, although products corresponding to cleavage at the \"a\" site were observed (Fig. 3.13c, lanes 2 and 4). Assays on rpsT mRNA are shown in Fig. 3.13d. Lanes 5 and 6, the positive controls, contain degradosomes. The final 147 nt product of RNase E processing is visible. RNase G and RNase G C405S/C408S showed similar patterns of cleavage of rpsT mRNA to degradosomes(Fig. 3.13d, compare lane 2 and lanes 3 and 4 to 5 and 6). Importantly, all three preparations produced the cleavage at site \"c\" (Fig. 3.13b) leading to an accumulation ofthe 147 nt product. A more detailed examination of the activity of the RNase G mutants was undertaken, with the results shown in Fig. 3.14 and Table 3.3. Activity was assayed using both monophosphorylated 671 nt and 728 nt substrates (see Fig. 2.1d, e) as well as full length 9S RNA (Fig. 2.1a). The relative activities of each RNase G variant were determined as described in Materials and Methods, section 2.8.1, with results graphed in Fig. 3.14a-c and compiled in Table 3.3. Although none ofthe cysteine to serine mutations resulted in a significant loss of activity, the various forms of RNase G could be sorted into two groups based on their rates of cleavage relative to wild-type RNase G (Table 3.3, line 1). RNase G-His6 and RNase G C405S/C408S form the first group. Both enzymes exhibited moderately increased rates of cleavage of reconstituted pre-16S rRNA substrates relative to wt RNase G, but reduced rates on 9S RNA (Table 2.3, 96 Figure 3.14. Endonuclease activity of RNase G variants with cysteine to serine mutations. Activities of RNase G preparations (see Table 2.2) on three model RNA substrates were determined. Each assay contained 0.6 pmoles RNA and approximately 18 pmoles RNase G preparation. Panel a shows the rate of disappearance of 5'-monophosphorylated 9S RNA substrate. The substrate is described in Fig. 2.1, and the assay is described in Materials and Methods, section 2.8.3. Panels b and c illustrate the rate of disappearance of the 728 nt (Fig. 2.1) and 671 nt (Fig. 2.1) RNP substrates (section 2.8.1). These data were used to construct Table 3.3, with the rates of each mutant on 9S determined by the time at which 75% of the substrate remained and the time at which 50% of the 728 and 671 nt substrates remained relative to wild-type RNase G. The data points for each mutant are as follows: wild type, untagged RNase G, +; His-tagged RNase G, •; RNase G C162S, •; RNase G C402S, *; RNase G C421S, A; RNase G C405S/C408S, • ; RNase G C402S/C405S/C408S, X. 97 98 lines 2,3). The RNase G variants in the second group (last four entries in Table 2.3), display reduced activities (20-60% of wt) on all three substrates. It is important to note that the rate of substrate disappearance was much lower for allthe RNase G preparations with 9S RNA as the substrate, and enzyme was used in excess over substrate to achieve observable activity. This agrees with earlier reports that found RNase G is incapable of simulating RNase E-mediated processing of 9S RNA when enzyme is not present in excess over substrate (Jiang et al., 2000; Tock et al., 2000). This excess of enzyme may be required if the turnover of the enzyme is slow and/or only a fraction of the preparation is active. The effect of oligomerization on the activity of different RNase G variants was examined. Fractions from the sucrose gradients in Fig. 3.12a were assayed for activity, including wt RNase G (fraction 13), RNase G-His6 (fraction 14), RNase G C405S/C408S (fraction 14) and RNase G C402S/C405S/C408S. The assay is depicted in Fig. 3.12b. It should be noted that an additional band, smaller than the intact substrate, but larger than the primary product (resulting from cleavage at the \"E\" site, Fig. 2.1), transiently accumulates. This product was observed when the 519 nt RNA was not pre-digested with RNase H and oligo GMV 2011, which mimics the RNase III cleavage (Fig. 2.1). This band is presumably due to accessibility of an additional cleavage site. Each fraction exhibited activity (Fig. 3.12b, compare lanes 3 to 6). Combining this data and the results discussed in section 3.2.4.2, two interpretations can be made. In the first, dimerization is not required for activity. Dimerization, rather, increases the rate 99 of endoribonucleolytic cleavage. In the second interpretation, dimerization is required for activity. RNase G may exist in a monomer/dimer equilibrium. With the active RNase G variants, RNase G-His6 and RNase G C405S/C408S, the equilibrium favours dimers. This results in a comparitivley high activity. The equilibrium for RNase G C402S/C405S/C408S, however, favours monomers. This results in a smaller proportion of the protein existing in the active, dimeric state, and lowered activity. 3.2.5. Effect ofS1 deletion on RNase G activity Both RNase E and RNase G contain an S1 domain. This potential RNA binding domain (reviewedby (Coburn and Mackie, 1999)) spans approximately residues 35 to 135 (see Fig. 3.1). Residues 36 to 135 were removed from RNase G (Chapter 2, section 2.4.3) to create a His-tagged protein lacking the S1 domain. RNase GAS1-His6 was purified as described in Chapter 2, section 2.6.3. Due to a lack of solubility, however, the final step in RNase GAS1-His6 purification was the Talon column. This yielded a pool of proteins enriched for RNase GAS1-His6, which accounted for approximately half of the protein in the sample. Successful introduction of the deletion was confirmed by sequencing and mass spectrometry (Table 3.2). Assays of RNase G-His6 and RNase GAS1-His6 with triphosphorylated 519 nt substrate (Fig. 2.1) were performed as described (Chapter 2, section 2.8.3). Products of the assay were separated on an 8% polyacrylamide gel, as shown in Fig. 3.15. Activity of RNase GAS1-His6 does not appear to be diminished, compared to RNase G-His6 (compare lanes 1-6 100 RNase G-His6 RNase GAS1-His6 0 5 15 30 45 60 0 5 15 30 45 60 min ^ ^ ^ ^ ^ . ! - » 1 1 f I I 1 2 3 4 5 6 7 8 9 10 11 12 Figure 3.15. Effect of S1 deletion on RNase G endoribonucleolytic activity. Assays combining 0.6 pmoles 3 2 P-labelled, triphosphorylated 519 nt substrate (Fig. 2.1) with 0.55 pmoles RNase G-His6 or RNase GAS1-His6 were performed as described in Chapter 2, section 2.8.3. Products were separated on an 8% polyacrylamide gel and visualized by Phosporimaging. Intact substrate (S) and product (P) are marked with arrows, and time points are indicated along the top ofthe gel. <4P 101 with 7-12) on triphosphorylated substrate. Both enzyme preparations yield the product (P) resulting from cleavage at the \"E\" site (Fig. 2.2), and RNase GAS1-His6 even accumulates products which appear to correspond to cleavage at the \"G\" site (band below \"P\" in lanes 10 through 12). The band just below intact substrate likely results from cleavage at a site present in the 519 nt substrate (see section 3.2.4.2). 3.2.6. rRNA processing activity of recombinant RNase G Examination of the processing of precursors to16S rRNA in vivo has shown that prior cleavage of rRNA precursors by RNase E facilitates the subsequent action of RNase G. RNase G, in turn, forms the mature 5'-end of 16S rRNA (Li et al., 1999b). Templates were prepared for the transcription of derivatives of pre-16S rRNA that would include the 5'-external transcribed sequence (5'-ETS) from the P2 promoter to the mature 5'-end (-173 nt) and the full 5'-domain of 16S rRNA (residues 1-556) as described in Materials and Methods (section 2.8.1) and shown in Figs. 2.1d and 3.16c. Ribonucleoprotein substrates, mimicking the in vivo target of RNase G, were produced by binding the RNA to either ribosomal protein S20 or S20 and S4 combined. To produce a substrate resembling the product of an initial RNase III cleavage, we used oligonucleotide GMV2011 to direct RNase H to cleave the RNA at or near residues -115/-116 (Fig. 3.16c; Fig 3.16a, compare lanes 1 and 2). This treatment also produces a 5'-monophosphorylated terminus on the resultant 519 nt RNA. 102 Figure 3.16. Reconstitution of RNP substrate processing. Panel a shows a 6% polyacrylamide urea-polyacrylamide gel of the processing of a 576 nt RNP. The 576 nt RNP is represented in Panel c. Experiments were performed as described in section 2.8.3, but the initial substrate was 0.6 pmoles tri-phosphorylated 576 nt RNA arising from transcription of pGM119 linearized with BstUI (see Fig. 2.1). Lanes 2-5 included oligonucleotide GMV2011 (indicated as a horizontal line above the -115 site in Panel c) in the heating step, and 1 U RNase H during the 30 min incubation at 30°C with ribosomal proteins S4 and S20. Lanes 4 and 5 include 18 pmoles RNase G (His-tagged) and lanes 3 and 5 contain 200 ng wild-type degradosomes from strain CF881. The arrows to the left of the panel indicate the position of the starting substrate (576 nt) and the 519 nt product that arises from the oligo-directed cleavage by RNase H (see above), which mimics the RNase III processing event (Panel c). Arrows to the right of the panel indicate the products arising from processing at the \"E\" ( -430-450 nt) and \"G\" (-375-400 nt) sites (Panel c). Panel b shows the kinetics of formation of the -375-400 nt product from the 519 nt RNA (\"precleaved\" at the RNase III site): •, RNase G alone; •, degradosomes alone; • , RNase G + degradosomes. This experiment was performed by G. A. Mackie. 103 + + + + RNase H - + - + D-somes - - + + RNase G 1 2 3 4 5 b +-> t > 700 3 -Q 600 O i- 500 D-tfl 400 ~ U - A g:g \\f G \" C . ft-U G A A u A U G A U - A G Q C G C - G 3 1 0 — A - U U - A C - G C - G G - C C - G C - G G - C G - C C - G C - G —360 A A A U - A G U U G U U G U C - G U \" A A G A A - U A C C G A G C U C G A A U U ^ I I I I I 210 300 | 350 370 RSR 126 Figure 4.5. PNPase activity of pnp13 degradosomes. PNPase activity was assessed for wild type and pnp13 degradosomes. Experiments are described in Coburn et al. (1999). Assays were performed as described in section 2.8.3, with the exceptions that the final assay volume was 40 pi; the reactions were carried out in the presence of 10 mM Na-phosphate and 3 mM ATP. A schematic of the substrate, malEF RNA, appears in Fig. 4.4. Processing products are indicated to the right of each panel. Panel a depicts the processing of malEF RNA by 200 ng/ml wild type, and Panel b depicts the processing of malEF RNA by 200 ng/ml pnp13 degradosomes. Panel c shows the products of processing of 200 ng/ml pnp13 degradosomes with 60 ng/ml PNPase added in trans. These assays were performed by Glen A. Coburn, and the figure is reprinted with permission from Coburn etai. (1999). 127 3 wt Degradosomes +ATP 0 2.5 5 10 15 20 30 45 60 (min) § § • „ ^ malEF m m m mm mm mm - ^ R S R pnp-13 Degradosomes +ATP 0 2.5 5 10 20 30 45 60 (min) 4 malEF pnp-13 Degradosomes +Pnp +ATP 0 1 2 5 10 20 30 45 60 (min) m *B m ~ - < ma/EF w w v -4 * RSR 128 due to RNase E activity. To determine if this lack of exonucleolytic processing could be complemented, purified wild type PNPase was added to the reaction in trans (Fig. 4.5c). Over the course of the assay, the first intermediate (*) accumulated transiently (Fig. 4.5. Panel a 2.5 and 5 min, and Panel c 1 and 2 min) and the RSR intermediate accumulated stably over the course of the reaction. In contrast to wild type degradosomes, however, this was the end product of the reaction, and complete degradation of the RSR intermediate was not observed. PNPase could not complement the pnp13 mutant in trans. It appears that PNPase must be coordinated with RhIB in degradosomes to activate RhIB, and that PNPase does not cycle on and off of the degradosome, since functional PNPase could not displace inactive pnp13 PNPase in the reaction. 4.2.4. Phosphatase activity of the degradosome During these studies of the degradosome-directed endonucleolytic cleavage of 5'-end labelled oligoribonucleotides (section 2.6.2), it was noted that label was lost during endoribonuclease assays. Originally, this was assumed to be due to exonucleolytic degradation of products. Isolation of pnp13 degradosomes, which lack PNPase activity (see above, section 4.2.3) was undertaken to remove exonucleolytic activity from the degradosome. Endoribonuclease assays (section 2.8.3) were performed with pnp13 degradosomes and 5'-end labelled oligonucleotide BR10 (see Fig. 5.1, 5.2). Products of the reaction are depicted in Fig. 4.6a, lanes 1-6. The amount of 3 2 P label remaining was determined by quantifying total label in the substrate (S; 10 nt) and product (P; 5 nt) bands by 129 Figure 4.6. Phosphatase activity of degradosomes. Degradosomes were analyzed for 5'-dephosphorylation activity. In Panel a, products arising from an endonuclease assay (section 2.8.3) containing 1 pg pnp13 degradosomes, 0.2 pM 5'-monophosphorylated BR10 RNA oligonucleotide substrate (see section 2.8.2 for substrate preparation, and Fig. 5.1 for BR10) and 0 (lanes 1-6), 5 mM (lanes 7-12) or 1 mM sodium fluoride (NaF; lanes 13-18). Separation was on a 15% polyacrylamide sequencing gel. Substrate (10 nt) and cleavage products (5 nt) are indicated to the right of the panel with \"S\" and \"P\" respectively. Remaining label was determined by summing the recoveries of substrate and product using a phosphorimager and ImageQuant software (Molecular Dynamics). These data are found in Panel b: •, 0 NaF; •, 5 mM NaF; A, 1 mM NaF. In Panel c, y32P-ATP (0.17 pCi/pl) was incubated with either 0.1 U/pl calf intestinal phosphatase or 400 ng pnp13 degradosomes as described in section 2.8.6. Products (8 pi) were then separated by thin layer chromatography on polyethyleneimine-impregnated plates in 0.375 M KH 2P0 4 (section 2.6.6). The origin is marked to the right by \"O\" and the products by \"P\". Lane 1 is the positive control, which followed incubation for 45 min in the presence of 0.1 U/pl calf intestinal phosphatase; lane 2 is the negative, time zero control and lane 3 contains the products resulting from incubation with 400 ng pnp13 degradosomes for 10 min (section 2.8.6). 130 a c 131 phosphorimaging and subsequent analysis using ImageQuant software (Molecular Dynamics). These data are plotted in Fig. 4.6b. Despite the lack of exonuclease activity, there was still significant loss of label over the course of the assay (Fig. 4.6b, •). The oligonucleotide substrate was labelled with 3 2 P at its 5'-end, and it was therefore possible that the loss of label was due to the action of a phosphatase. Since RNase E is 5'-end dependent (Mackie, 1998), it was possible that RNase E removed the 5'-phosphate. Phosphatase activity is inhibited by the addition of sodium fluoride (NaF)(Gilboe and Nuttall, 1976; Yeaman and Cohen, 1975), so endonuclease digestions were performed in the presence of 5 and 1 mM sodium fluoride (Fig. 4.6a, lanes 7-12 and 13-18, respectively). Comparing these to the control, it was noted that addition of NaF did not inhibit the loss of label. It was, therefore, unlikely that RNase E was behaving as a phosphatase. This was confirmed by comparing the products of Y32P-ATP treated with calf intestinal alkaline phosphatase (New England Biolabs) or pnp13 degradosomes by thin layer chromatography (Fig. 4.6c and section 2.8.6). Fig. 4.6c lane 1 is the positive control, and it shows the liberation of free phosphate (P) by calf intestinal alkaline phosphatase. Fig. 4.6c, lane 2 is the time zero negative control, and two spots are visible. The first is intact v3 2P-ATP, and is located at the origin (O), and the second is a faint band just below free phosphate (P). This second band was also observed with thin layer chromatography of products from the reaction between BR10 and degradosomes, as shown in Fig. 4.7c, lanes 6-10. Importantly, this second band is also present in the negative control (Fig. 4.6c lane 2) that contained degradosomes and y32P-ATP, but lacked any incubation time. This second band 132 is therefore likely a contaminant of the y P-ATP and the loss of label is not due to degradosome-mediated phosphatase activity liberating free phosphate from Y32P-ATP. The source of the contaminating band found in Fig. 4.6c lanes 2 and 3 was sought by precipitating the oligonucleotides following labelling, but prior to performing the endoribonuclease assay (section 2.8.2). Fig. 4.7a confirmed that precipitation had no effect on the products of the endonuclease assays (Fig. 4.7a, compare lanes 1-5 and 6-10). Quantification of the amount of label remaining (Fig. 4.6b) revealed that precipitation also had no effect on the loss of 5'-label, as expected (compare unprecipitated, \"•\" and precipitated \"A\"). Assay products were then separated by thin layer chromatography (Fig. 4.7c). Precipitation of the oligonucleotide (Fig. 4.7c lanes 1-5) removed the contaminating band, which is still visible in lanes 6-10, where the oligonucleotides were not precipitated prior to assay. By comparing Fig. 4.7c lanes 6-10 and Fig. 4.6c lane 2 (y32P-ATP) it appears that a radioactive contaminant was present in the labelled ATP. Possible explanations for the loss of label will be described in the Discussion, section 4.3. 4.3. Discussion Degradosomes were successfully isolated from E. coli strain RD100, which carries the pnp13 allele that leads to the inactivation of PNPase (Reiner, 1969). The composition of these degradosomes was equivalent to those isolated from strain CF881 \"wild type\" (Fig. 4.1 lanes 2 and 3). The endonucleolytic activity of 133 Figure 4.7. Phosphatase activity of pnp13 degradosomes on ethanol-precipitated oligoribonucleotides. Panel a is a 15% sequencing gel of the products arising from endonuclease assays (section 2.8.3) containing 0.4 pM BR13 (see Fig. 5.1) and 50 ng pnp13 degradosomes. In lanes 1-5 (ppte), BR13 was precipitated with ethanol (section 2.8.2) prior to assaying while the BR13 in lanes 6 through 10 (unppte) was not precipitated. Intact substrate (S) and products (P) are indicated to the right of the panel. As with Fig. 4.5, total label remaining was calculated for each time point in Panel a, and graphed in Panel b. Data points for precipitated samples (A) and unprecipitated (•) samples are as indicated. Assay samples were also prepared and separated by thin layer chromatography using polyethyleneimine impregnated plates and K 2HP0 4 as the solvent (section 2.8.6). The direction of migration is indicated with an arrow. Each lane contains 5 pi of sample, with lanes 1 - 5 containing samples from assays using precipitated BR13, and lanes 6-10 samples using unprecipitated BR13. 134 Precipitation c Precipitation 0 5 10 15 30 0 5 10 15 30 min + i I I 1 0 5 101530 0 5 10 1530 min 1 2 3 4 5 6 7 8 9 10 b O) c c 100 'co E CD i— 75 \"55 n 1 2 3 4 Ms IP I pre-5S 8 9 0 30 60 0 30 60 0 30 fin min Hfe mm. mm, mmt *m+ mm mm mm Mm* ^mw mm mr 1 2 3 ^S pre-5S 8 9 100 Dl C re E £ o E •»-» 05 .C ~s 100 _£ '£ 're E 5 CA .Q 3 3) HO-DNA 2) P-DNA 1) no competitor 3) HO-DNA 2) P-DNA no competitor Time (min) Time (min) 163 which is in turn based on the 5'-end of RNA 1 (Fig. 5.1 and 5.2)(McDowall et al., 1995). From these data, it was determined that 5'-OH DNA oligonucleotides are very strong inhibitors of RNase E/G activity. Similar inhibition was observed with all DNA oligonucleotides examined, and inhibition was observed with as little as a three-fold excess of inhibitor to enzyme (data not shown). Further experiments are required to determine the mode and efficiency of inhibition. If these oligonucleotides are established as strong inhibitors of RNase E/G activity, they may aid in crystallographic efforts and/or illuminating the active site or nucleotide binding regions. In the course of this thesis, some of the mysteries concerning the structure and specificity of RNase E and RNase G were solved. By establishing RNase G as a model for RNase E activity, the groundwork for future projects has been laid. In the short term, the focus should be on mutating highly conserved residues in RNase G and determining their effects on endonucleolytic processing. 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Each lane contains 7 pi of a collected fraction separated on a 10% SDS-polyacrylamide gel (section 2.7.1). Proteins were visualized by staining with Coomassie Brilliant Blue R-250. The concentration of NaCl in the running buffer increases from left to right. Panel a shows the collected fractions from the Heparin-agarose column. Panel b shows fractions from the anion exchange column. The lanes containing molecular weight standards are indicated on both gels. The size of each standard is indicated to the left of the panel. 187 188 Appendix Figure 2. Gel filtration size determination of RNase G samples. Samples were eluted in 25 mM Tris-HCl pH 7.6, 320 mM NaCl on a calibrated Superdex S200 column as described in Materials and Methods, section 2.7.8. The volume of injection for each sample is marked by \"t\", and the volumes of the corresponding peaks for each sample are indicated above each peak. Volumes were determined using Unicorn software (Pharmacia). 189 190 191 "@en ; edm:hasType "Thesis/Dissertation"@en ; vivo:dateIssued "2003-11"@en ; edm:isShownAt "10.14288/1.0091231"@en ; dcterms:language "eng"@en ; ns0:degreeDiscipline "Biochemistry and Molecular Biology"@en ; edm:provider "Vancouver : University of British Columbia Library"@en ; dcterms:publisher "University of British Columbia"@en ; dcterms:rights "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en ; ns0:scholarLevel "Graduate"@en ; dcterms:title "Functions and properties of RNase G and RNase E from Escherichia coli"@en ; dcterms:type "Text"@en ; ns0:identifierURI "http://hdl.handle.net/2429/15021"@en .