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• W2009608230 abstract "In eubacteria, the ς subunit binds to the core RNA polymerase and directs transcription initiation from any of its cognate set of promoters. Previously, our laboratory defined a region of the β′ subunit that interacts with ς70 in vitro. This region of β′ contained heptad repeat motifs indicative of coiled coils. In this work, we used 10 single point mutations of the predicted coiled coils, located within residues 260–309 of β′, to look at disruption of the ς70-core interaction. Several of the mutants were defective for binding ς70 in vitro. Of these mutants, three (R275Q, E295K, and A302D) caused cells to be inviable in an in vivoassay in which the mutant β′ is the sole source of β′ subunit for the cell. All of the mutants were able to assemble into the core enzyme; however, R275Q, E295K, A302D were defective for Eς70 holoenzyme formation. Several of the mutants were also defective for holoenzyme assembly with various minor ς factors. In the recently published crystal structure of Thermus aquaticus core RNA polymerase (Zhang, G., Campbell, E. A., Minakhin, L., Richter, C., Severinov, K., and Darst, S. A. (1999)Cell98, 811–824), the region homologous to β′260–309 of Escherichia coli forms a coiled coil. Modeling of our mutations onto that coiled coil places the most defective mutations on one face of the coiled coil. In eubacteria, the ς subunit binds to the core RNA polymerase and directs transcription initiation from any of its cognate set of promoters. Previously, our laboratory defined a region of the β′ subunit that interacts with ς70 in vitro. This region of β′ contained heptad repeat motifs indicative of coiled coils. In this work, we used 10 single point mutations of the predicted coiled coils, located within residues 260–309 of β′, to look at disruption of the ς70-core interaction. Several of the mutants were defective for binding ς70 in vitro. Of these mutants, three (R275Q, E295K, and A302D) caused cells to be inviable in an in vivoassay in which the mutant β′ is the sole source of β′ subunit for the cell. All of the mutants were able to assemble into the core enzyme; however, R275Q, E295K, A302D were defective for Eς70 holoenzyme formation. Several of the mutants were also defective for holoenzyme assembly with various minor ς factors. In the recently published crystal structure of Thermus aquaticus core RNA polymerase (Zhang, G., Campbell, E. A., Minakhin, L., Richter, C., Severinov, K., and Darst, S. A. (1999)Cell98, 811–824), the region homologous to β′260–309 of Escherichia coli forms a coiled coil. Modeling of our mutations onto that coiled coil places the most defective mutations on one face of the coiled coil. RNA polymerase nickel nitrilotriacetic acid wild type The DNA-dependent RNA polymerase (RNAP)1 is a multisubunit enzyme that plays a central role in eubacterial gene regulation and expression. This enzyme has two functional forms: core and holoenzyme. Transcription elongation and termination are performed by the core enzyme with α:2,β:1,β′:1 stoichiometry (1Helmann J.D. Chamberlin M.J. Annu. Rev. Biochem. 1988; 57: 839-872Crossref PubMed Scopus (716) Google Scholar). Core RNAP binds one of a variety of ς subunit species at a given time to form a specific holoenzyme (2Burgess R.R. Travers A.A. Dunn J.J. Bautz E.K.F. Nature. 1969; 221: 43-46Crossref PubMed Scopus (639) Google Scholar). The holoenzyme performs the tasks of promoter recognition and transcription initiation. Each ς subunit directs its cognate holoenzyme to start transcription from only those promoters containing DNA sequences specifically recognized by that ς factor (2Burgess R.R. Travers A.A. Dunn J.J. Bautz E.K.F. Nature. 1969; 221: 43-46Crossref PubMed Scopus (639) Google Scholar, 3Gross C.A. Chan C.L. Lonetto M.A. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 1996; 351: 475-482Crossref PubMed Scopus (36) Google Scholar, 4Gross C.A. Lonetto M. Losick R. McKnight S. Yamamoto K. Transcriptional Regulation. Cold Spring Harbor Press, Cold Spring Harbor, NY1992: 129-176Google Scholar). Understanding the molecular basis of ς binding to core RNAP will aid in the analysis of several questions involving how multiple ς species come to sequester core and turn on subsequent genes. It has been hypothesized that all of the ς species bind to the same site/sites on the core enzyme (1Helmann J.D. Chamberlin M.J. Annu. Rev. Biochem. 1988; 57: 839-872Crossref PubMed Scopus (716) Google Scholar, 6Sharp M.M. Chan C.L. Lu C.Z. Marr M.T. Nechaev S. Merritt E.W. Severinov K. Roberts J.W. Gross C.A. Genes Dev. 1999; 13: 3015-3026Crossref PubMed Scopus (133) Google Scholar, 7Traviglia S.L. Datwyler S.A. Yan D. Ishihama A. Meares C.F. Biochemistry. 1999; 38: 15774-15778Crossref PubMed Scopus (38) Google Scholar). Studies to identify the core binding site on ς have resulted in the positive identification of a single binding site located on ς70 overlapping conserved region 2.1 (5Lonetto M. Gribskov M. Gross C.A. J. Bacteriol. 1992; 174: 3843-3849Crossref PubMed Scopus (737) Google Scholar,8Lesley S.A. Burgess R.R. Biochemistry. 1989; 28: 7728-7734Crossref PubMed Scopus (109) Google Scholar). A single point mutation in the homologous region of Bacillus subtilis ςE prevented binding to core (9Shuler M.F. Tatti K.M. Wade K.H. Moran Jr., C.P. J. Bacteriol. 1995; 177: 3687-3694Crossref PubMed Google Scholar). More recent genetic and biochemical studies suggest that region 2.1 may be only one of multiple contact sites that ς uses in binding to the core enzyme (6Sharp M.M. Chan C.L. Lu C.Z. Marr M.T. Nechaev S. Merritt E.W. Severinov K. Roberts J.W. Gross C.A. Genes Dev. 1999; 13: 3015-3026Crossref PubMed Scopus (133) Google Scholar, 10Joo D.M. Nolte A. Calendar R. Zhou Y.N. Jin D.J. J. Bacteriol. 1998; 180: 1095-1102Crossref PubMed Google Scholar, 11Nagai H. Shimamoto N. Genes Cells. 1997; 2: 725-734Crossref PubMed Scopus (45) Google Scholar). Information about sites on core that bind ς has come in most part from biochemical assays. Protein footprinting studies of the core enzymes susceptibility to hydroxy radical cleavage upon binding a modified ς factor containing the cleavage catalyst have revealed that there are three regions on the core, one located on β′ and two on β, that are in close proximity to ς (12Owens J.T. Miyake R. Murakami K. Chmura A.J. Fujita N. Ishihama A. Meares C.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6021-6026Crossref PubMed Scopus (86) Google Scholar). One of the β regions was identified earlier as being near the ς70 binding region. It was noticed that this site, in the core enzyme complex, was cleaved by trypsin, whereas formation of Eς70 prevented trypsin cleavage at this site (13Fisher R. Blumenthal T. J. Biol. Chem. 1980; 255: 11056-11062Abstract Full Text PDF PubMed Google Scholar). Previous in vitro work from our laboratory identified a strong binding site for ς70 on the β′ subunit (14Arthur T.M. Burgess R.R. J. Biol. Chem. 1998; 273: 31381-31387Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). This site was mapped to within residues 260–309 of β′. The predicted secondary structure (15Rost B. Sander C. Schneider R. CABIO S. 1994; 10: 53-60PubMed Google Scholar) of β′260–309 has two α helices joined by a random coil. Another structural analysis program indicates that these two helices are amphipathic and have the potential for coiled coil formation (16Lupas A. Van Dyke M. Stock J. Science. 1991; 252: 1162-1164Crossref PubMed Scopus (3482) Google Scholar). The coiled coil motif is based on a heptad repeat of residues designated a–g (17Cohen C. Parry D.A.D. Trends Biochem. Sci. 1986; 11: 245-248Abstract Full Text PDF Scopus (302) Google Scholar, 18Chao H. Bautista D.L. Litowski J. Irvin R.T. Hodges R.S. J. Chromatogr. B Biomed. Sci. Appl. 1998; 715: 307-329Crossref PubMed Scopus (90) Google Scholar) (Fig.1 B). The a and d positions are hydrophobic, whereas the other positions are usually charged or polar. Burial of the a and d hydrophobic residues during coiled coil formation provides a large amount of the binding energy. Specificity in binding comes from the e and g positions, which can form ionic interactions or salt bridges. We have undertaken a mutational analysis of this region to confirm that our in vitro binding results were relevant to in vivo binding and function. This work presents the analysis of 10 point mutations, most of which are change-of-charge mutations at the e and g residues, in the β′260–309 predicted coiled coil. Three of the mutations (R275Q, E295K, and A302D) were nonfunctional in binding ς70 in all of the assays in which they were tested but still able to assemble into the core enzyme. We also report on mutations that were nonfunctional in some of our assays but functional in others, indicating that binding of other sites may compensate for loss of binding at the β′260–309 site. We use this analysis to demonstrate that the binding site identified previously by in vitro methods is important in vivo and that mutations in this region can greatly diminish core binding of ς70 and other minor ςs. In the recently solved crystal structure for the core RNAP of Thermus aquaticus, the region homologous to Escherichia coliβ′260–309 was determined to form a “coiled coil-like” structure (19Zhang G. Campbell E.A. Minakhin L. Richter C. Severinov K. Darst S.A. Cell. 1999; 98: 811-824Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar) consistent with our predictions. Modeling of our mutations onto the T. aquaticus structure places all of the nonfunctional mutations on the same face of the β′ coiled coil. Plasmid characteristics are described in Table I. Plasmids pTA577 and 600–620 were made from the base plasmid pRL663 (20Wang D. Meier T.I. Chan C.L. Feng G. Lee D.N. Landick R.L. Cell. 1995; 81 (350): 341Abstract Full Text PDF PubMed Scopus (120) Google Scholar). SingleHinDIII and BamHI restriction sites, at bases 674 and 952 of rpoC, respectively, were inserted into the rpoC gene of pRL663 via silent mutagenesis to create pTA577. pTA561 was created in the same manner as pTA577 except that pRL308 (22Weilbaecher R. Hebron C. Feng G. Landick R. Genes Dev. 1994; 8: 2913-2917Crossref PubMed Scopus (82) Google Scholar) was the starting plasmid. The HinDIII and BamHI restriction sites were used to insert polymerase chain reaction-generated DNA fragments containing the various mutations to generate pTA600–609. For pTA620, which contains a truncatedrpoC fragment coding for β′ residues 1–319, pRL663 was cut with Xba-HinDIII for insertion of a polymerase chain reaction-generated rpoC truncation. The ς70binding site was previously mapped to residues 260–309 of β′; however, we engineered some of the constructs for this work to extend to residue 319. This was done to incorporate the BamHI site mentioned previously. Therefore, the various mutations could be moved into the new plasmid to create pTA610–619. We have not seen any difference in behavior of the fragments ending at residue 309 as opposed to those ending at residue 319. All sequences generated via polymerase chain reaction were sequenced to ensure that spurious mutations had not been incorporated.Table IPlasmid list and characteristicsPlasmidβ′ residuesMutationsModificationsRef.pRL3081–1407NoneNone22Weilbaecher R. Hebron C. Feng G. Landick R. Genes Dev. 1994; 8: 2913-2917Crossref PubMed Scopus (82) Google ScholarpRL6631–1407NoneC-terminal His620Wang D. Meier T.I. Chan C.L. Feng G. Lee D.N. Landick R.L. Cell. 1995; 81 (350): 341Abstract Full Text PDF PubMed Scopus (120) Google ScholarpTA5611–1407SilentNoneThis workpTA5771–1407SilentC-terminal His6This workpTA6001–1407N266DC-terminal His6This workpTA6011–1407Y269AC-terminal His6This workpTA6021–1407R275QC-terminal His6This workpTA6031–1407K280EC-terminal His6This workpTA6041–1407R293QC-terminal His6This workpTA6051–1407E295KC-terminal His6This workpTA6061–1407R297SC-terminal His6This workpTA6071–1407Q300EC-terminal His6This workpTA6081–1407A302DC-terminal His6This workpTA6091–1407N309DC-terminal His6This workpTA6101–319N266DNoneThis workpTA6111–319Y269ANoneThis workpTA6121–319R275QNoneThis workpTA6131–319K280ENoneThis workpTA6141–319R293QNoneThis workpTA6151–319E295KNoneThis workpTA6161–319R297SNoneThis workpTA6171–319Q300ENoneThis workpTA6181–319A302DNoneThis workpTA6191–319N309DNoneThis workpTA6201–319SilentNoneThis work Open table in a new tab The cells were grown to an A 600 of 0.6–0.8 in 1-liter cultures at 37 °C in LB medium with 100 μg/ml ampicillin. Isopropyl-β-d-thiogalactopyranoside was then added to a concentration of 1 mm. Three hours after induction, the cells were harvested by centrifugation at 8,000 × gfor 15 min and frozen at –20 °C. The cell pellet from a 1-liter culture was thawed and resuspended in 10 ml of lysis buffer (40 mm Tris, pH 7.9, 0.3 mKCl, 10 mm EDTA, and 0.1 mmphenylmethylsulfonyl fluoride), and lysozyme was added to 0.1 mg/ml. The cells were incubated on ice for 15 min and then sonicated in three 60-s bursts. The recombinant protein in the form of inclusion bodies was separated from the soluble lysate by centrifugation at 27,000 × g for 15 min. The inclusion body pellet was resuspended by sonication in 10 ml of lysis buffer + 2% (w/v) sodium deoxycholate. The mixture was centrifuged at 27,000 × g for 15 min, and the supernatant was discarded. The deoxycholate-washed inclusion bodies were resuspended in 10 ml of deionized water and centrifuged at 27,000 × g for 15 min. The water wash was repeated, and the inclusion bodies were aliquoted into 1-mg pellets and frozen at −20 °C until use. ς70 inclusion bodies (10 mg) were solubilized, refolded, and purified according to a variation of the procedure of Gribskov and Burgess (21Gribskov M. Burgess R.R. Nucleic Acids Res. 1986; 14: 6745-6763Crossref PubMed Scopus (342) Google Scholar). The inclusion bodies were solubilized by resuspension in 10 ml of 6 m guanidine-HCl. The proteins were allowed to refold by diluting the denaturant 64-fold with Buffer A (50 mm Tris, pH 7.9, 0.5 mm EDTA, and 5% (v/v) glycerol) in 2-fold steps over 2 h. One gram of resin (DEAE-cellulose, Whatman) was added and mixed with slow stirring for 24 h at 4 °C. The resin was then collected in a 10-ml column and washed, and the protein was eluted with a gradient from 0.1 to 1.0m NaCl in Buffer A. The ς70 fractions were pooled, dialyzed overnight against 1 liter of storage buffer (50 mm Tris, pH 7.9, 0.5 mm EDTA, 0.1 mNaCl, 0.1 mm dithiothreitol, and 50% (v/v) glycerol), and stored at −20 °C. Protein samples to be quantitated were subjected to SDS-polyacrylamide gel electrophoresis. The proteins were electrophoretically transferred out of the gel onto 0.05-μm nitrocellulose. The blot was blocked in Blotto (10 mm Tris, pH 7.4, 150 mm NaCl, 0.1% (v/v) Tween 20, and 3% (w/v) nonfat dry milk) and probed with monoclonal antibodies. The signal was generated using the ECL Plus system (Amersham Pharmacia Biotech) and detected on a Storm PhosphorImager (Molecular Dynamics). The signal was quantitated using ImageQuant software (Molecular Dynamics). Cells containing truncated β′ expression plasmids pTA610–620 were grown to A 600 = 0.6–0.8 and induced with 1 mm isopropyl-β-d-thiogalactopyranoside. The cells were grown for an additional 30 min. A 200-μl sample was removed and sonicated three times for 30 s each. 20 μl of glycerol and 20 μl of SDS sample buffer were added and heated for 2 min at 95 °C, and the sample was then stored at –20 °C until use. The lysates were separated by SDS-polyacrylamide gel electrophoresis. The proteins were electrophoretically transferred onto 0.05-μm nitrocellulose. The nitrocellulose was blocked by incubating in HYB buffer (20 mm Hepes, pH 7.2, 200 mm KCl, 2 mm MgCl2, 0.1 μmZnCl2, 1 mm dithiothreitol, 0.5% (v/v) Tween 20, 1% (w/v) nonfat dry milk) for 16 h at 4 °C. A chimeric ς70 was created by fusing a heart muscle kinase recognition sequence to the N terminus of ς70 to facilitate radiolabeling of ς70. Labeling of ς70 was done in a 100-μl reaction volume. 50 μl of 2× kinase buffer (40 mm Tris, pH 7.4, 200 mmNaCl, 24 mm MgCl2, 2 mmdithiothreitol) was added to 50 μg of heart muscle kinase ς70 protein. 240 units of cAMP-dependent kinase catalytic subunit (Promega) was added and the total volume was brought to 99 μl with deionized water. One microliter of [γ-32P]ATP (0.15 mCi/μl) was added. The mixture was incubated at room temperature for 30 min. The reaction mixture was then loaded onto a Biospin-P6 column (Bio-Rad) preequilibrated with 1× kinase buffer and centrifuged at 1100 × g for 4 min. The flow-through was collected and stored at −20 °C. The blocked nitrocellulose was incubated in 10 ml of HYB buffer with 4 × 105 cpm/ml 32P-labeled ς70 for 3 h at room temperature. The blot was washed three times with 10 ml of HYB buffer for 3 min each. The blot was dried, and the signal was visualized with a PhosphorImager and quantitated with ImageQuant software (Molecular Dynamics). Plasmids pTA577 and 600–609 (0.1 μg) were transformed into strain RL602 (22Weilbaecher R. Hebron C. Feng G. Landick R. Genes Dev. 1994; 8: 2913-2917Crossref PubMed Scopus (82) Google Scholar, 23Ridley S.P. Oeshger M.P. J. Bacteriol. 1982; 152: 736-746PubMed Google Scholar). After heat shock and incubation on ice, 300 μl of LB was added to the 50-μl cell mixture. 10 μl of the transformation reaction was spotted onto LB plates plus ampicillin (100 μg/ml) and incubated at 30 °C. Another 10 μl was spotted onto identical plates and incubated at 42 °C. The plates were incubated for 24–48 h and assessed for growth. 1-liter flasks containing 200 ml of LB with ampicillin (100 μg/ml) and isopropyl-β-d-thiogalactopyranoside (0.15 mm) were inoculated with 200 μl from overnight cultures of cells containing plasmids pTA561, 577, and 600–609. The cultures were grown at 37 °C with shaking until the A 600 reached 0.4 for log phase assays and 2 h longer (A 600 ∼ 2.0) for the early stationary phase assays. The cells were harvested by centrifugation at 6,000 × g for 10 min and stored at –20 °C until use. The cell pellets were resuspended in 5 ml of TE (10 mm Tris, pH 7.9, and 0.1 mm EDTA) plus 0.15 m NaCl and lysozyme (0.1 mg/ml) and then incubated on ice for 15 min. The cells were sonicated three times for 30 s each and centrifuged for 25 min at 27,000 × g to remove the insoluble material. The supernatant was loaded onto a 1.5-ml immunoaffinity column containing the polyol-responsive, anti-β′ monoclonal antibody NT73 (24Thompson N.E. Hager D.A. Burgess R.R. Biochemistry. 1992; 31: 7003-7008Crossref PubMed Scopus (70) Google Scholar). The column was washed with 15 ml of TE plus 0.15 m NaCl followed by a second wash with 10 ml of TE plus 0.5 m NaCl. The protein was eluted from the column with 4 ml of TE plus 0.7m NaCl and 30% propylene glycol. The eluted sample (4 ml) was diluted with 6 ml of buffer 66 (20 mm Tris, pH 7.9, 500 mm NaCl, 5 mm imidazole, 0.1% (v/v) Tween 20, and 10% (v/v) glycerol) and loaded (2×) onto 500 μl of Ni2+-NTA resin. The resin was washed twice with 5 ml of buffer 66 and eluted with 0.5 ml of buffer 66 plus 0.25 mimidazole. Samples from the elution fractions were assayed by Western blot as described above, using monoclonal antibodies to each subunit or ς factor. The secondary antibodies were horseradish peroxidase-labeled goat anti-mouse IgG antibodies and the signal was generated using the ECL Plus substrate system (Amersham Pharmacia Biotech) and detected using the STORM PhosphorImager and quantitated with ImageQuant software (Molecular Dynamics). Structural prediction, using the Coils program (16Lupas A. Van Dyke M. Stock J. Science. 1991; 252: 1162-1164Crossref PubMed Scopus (3482) Google Scholar), scored both of the predicted α-helices of β′260–309 as having a high probability of forming coiled coils (Fig. 1 A). To test this prediction, we constructed two β′ mutants with proline residues inserted into either helix. These β′ mutants were no longer predicted to form helices or coiled coils. When assayed for function in both the far Western and in vivo growth assays, both mutants were found to be nonfunctional (data not shown). We took this to indicate that the helical/coiled coil structure in this region was important for function. The solubility of these mutant proteins was not 100%, so we ceased using them because their loss of function could simply be due to gross folding defects. We decided to concentrate in most part on the e and g positions of the α-helices for the next phase of our analysis. The e and g residues of coiled coils often engage in interhelical interactions, such as the formation of ionic interactions or salt bridges (17Cohen C. Parry D.A.D. Trends Biochem. Sci. 1986; 11: 245-248Abstract Full Text PDF Scopus (302) Google Scholar, 18Chao H. Bautista D.L. Litowski J. Irvin R.T. Hodges R.S. J. Chromatogr. B Biomed. Sci. Appl. 1998; 715: 307-329Crossref PubMed Scopus (90) Google Scholar). Such interactions in this case could be intramolecular (between the two helices of β′260–309), forming a coiled coil structure necessary for binding by the ς subunit (Fig. 1 B). Alternatively, the e and g residues of β′260–309 could be making intermolecular contacts with ς upon binding. Our efforts were directed toward making change-of-charge mutations at these residues of β′ (Fig.1 B) and assaying their effects on binding. Two of the mutations described in this work do not involve e or g residues and were chosen for other reasons. Based on the findings that tyrosine and arginine residues are often located in “hot spots” of protein-protein interactions (26Bogan A.A. Thorn K.S. J. Mol. Biol. 1998; 280: 1-9Crossref PubMed Scopus (1638) Google Scholar), we changed the tyrosine residue at position 269 to an alanine and arginine 297 to a serine. Previous studies in our laboratory found that insertion of a leucine at position 297 generated a β′ subunit that was nonfunctional for binding ς70 (unpublished results). Therefore, we were interested to determine whether a less drastic mutation at this position would also affect ς binding. Previously, we had used far Western blotting to map a ς70 binding site to the N-terminal region of the β′ subunit (14Arthur T.M. Burgess R.R. J. Biol. Chem. 1998; 273: 31381-31387Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). We again applied this procedure as an initial assay for functionality of our β′ mutants. The mutations were cloned into a gene fragment coding for amino acids 1–319 of the β′ subunit. Cells containing these genes were induced for a short period to give moderate levels of the β′ fragment, comparable to other proteins in the extract. Samples were analyzed for binding ς70 by far Western analysis as described under “Experimental Procedures.” The amount of ς70 probe bound by each β′1–319mutant fragment was compared with the amount bound by wt β′1–319 fragment. Each signal was normalized to the amount of β′1–319 contained in the supernatant as determined by Western blotting. Five of the mutations (R275Q, R293Q, E295K, R297S, and A302D) were greatly reduced in their ability to bind ς70 (Fig.2). The Q300E and N309D mutations had the opposite effect, binding more ς70 than wild type β′1–319. Q300E exhibited an increase in relative binding of greater than 7-fold. There were no effects on binding seen with the N266D, Y269A, or K280E mutations. To assess the importance of the ς70 binding site in vivo, we assayed the ability of mutant β′ subunits to function as the sole source of β′ for the cell. Plasmids containing mutant or wild type, full-length β′ were transformed into strain RL602 (22Weilbaecher R. Hebron C. Feng G. Landick R. Genes Dev. 1994; 8: 2913-2917Crossref PubMed Scopus (82) Google Scholar, 23Ridley S.P. Oeshger M.P. J. Bacteriol. 1982; 152: 736-746PubMed Google Scholar). The chromosomal rpoC gene of RL602 has an amber mutation that prevents functional β′ from being produced in the absence of a suppressor tRNA. RL602 also has a chromosomal, temperature-sensitive amber suppressor. At the permissive temperature (30 °C), the amber suppressor is active and allows chromosomal β′ to be produced and the cell can grow. The amber suppressor is not active at the nonpermissive temperature (42 °C). Therefore, at 42 °C, chromosomal β′ is not made, and the cell cannot grow without another source of β′. If the plasmid-derived β′ can complement the loss of β′, then the cells will grow and form colonies on plates at the nonpermissive temperature. If the mutant β′ cannot complement, there will be no growth on the plates at this temperature. Three of the β′ mutants that were defective for ς binding in the far Western assay (R275Q, E295K, and A302D) could not support growth at the nonpermissive temperature, indicating that these mutations were also caused defects in binding ς in vivo (Fig.3). N266D, a mutation that had no detectable effect in the far Western assay, allowed some growth at the nonpermissive temperature but not enough to be considered wild type. In contrast, the R293Q and R297S mutations that did not bind ς70 in the far Western assay could support growthin vivo. Mutations Y269A, K280E, Q300E, and N309D had no detectable effects on growth. Expression levels for nonfunctional β′ mutants were determined to be equivalent to that of plasmid-derived, wild type β′ when grown at 37 °C (data not shown). An alternate explanation for the inviability caused by some of the β′ mutations would be that they are no longer able to be assembled into the core enzyme. To evaluate the potential assembly defects caused by the various mutations, we expressed His6-tagged, mutant β′ subunits in cells that were also expressing wild type, chromosomal β′ proteins. We used a Ni2+-NTA mediated pull-out assay to purify the mutant β subunits together with associated cell proteins. An immunoaffinity column was used to clean up the samples in order to reduce any nonspecific binding to the Ni2+-NTA column. All of the mutant β′ subunits tested retained the ability to assemble into the core enzyme demonstrated by the association of the α and β subunits throughout the purification (Fig.4, A and B). Again, mutations R275Q, E295K, and A302D caused defects in binding ς70 in both log and stationary phase samples (Fig.4 C). Also reduced in Eς70 formation were N266D in both log and stationary phase samples and R297S in log phase samples. Q300E again showed properties of binding ς70better than wild type. Y269A, K280E, R293Q, and N309D had no detectable effect on Eς70 assembly. When a non-His6-tagged β′ was expressed from the plasmid, there was no detectable nonspecific binding to the Ni2+-NTA column. All of the sample eluates were also assayed for the presence of any minor ς species. The only minor ςs of which the concentrations were sufficient for detection were ς32 in log phase and ς32 and ςF in stationary phase samples. The results for these ςs were essentially the same as for ς70 with the exception of mutants R297S and Q300E. In stationary phase samples from the Q300E mutant, the ς32and ςF levels are greatly reduced, whereas the ς70 levels are above those in the wt. The log phase samples for this mutant also contained a decreased amount of ς32 indicating a defect in Eς32 formation but not as severe as in stationary phase. Recently, Zhang et al. (19Zhang G. Campbell E.A. Minakhin L. Richter C. Severinov K. Darst S.A. Cell. 1999; 98: 811-824Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar) published the crystal structure of T. aquaticus core RNAP. The β′260–309 region of E. coli RNAP has a high degree of sequence conservation with its T. aquaticus homolog (Fig.5 A). 2L. Minakhin and K. Severinov, personal communication. This region of the T. aquaticus β′ subunit forms a coiled coil-like structure. When the mutations studied here are modeled onto the T. aquaticus structure using the Rasmol program (25Sayle R.A. Milner-White E.J. Trends Biochem. Sci. 1995; 20: 374Abstract Full Text PDF PubMed Scopus (2323) Google Scholar), those that are most defective in ς binding are grouped on one face of the coiled coil. Those that had defective phenotypes in some assays but not others are on the outer edges of this face. Mutations that had no detectable effects are clustered on the opposite face of the coiled coil, with the exception of N309D, which is located at the very C terminus of the coiled coil immediately next to the “rudder” (19Zhang G. Campbell E.A. Minakhin L. Richter C. Severinov K. Darst S.A. Cell. 1999; 98: 811-824Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar) (Fig. 5, B and C). Binding of various ς factors to the core polymerase is a major step in the process of global gene expression and regulation. It is not known whether this step is part of the regulation, via a competition for binding to a limited core population, or merely a straight-forward binding of free ςs to an excess of core (27Jishage M. Iwata A. Ueda S. Ishihama A. J. Bacteriol. 1996; 178: 5447-5451Crossref PubMed Google Scholar, 28Ju J. Mitchell T. Peters III, H. Haldenwang W.G. J. Bacteriol. 1999; 181: 4969-4977Crossref PubMed Google Scholar, 29Farewell A. Kvint K. Nystrom T. Mol. Microbiol. 1998; 29: 1039-1051Crossref PubMed Scopus (227) Google Scholar, 30Fujita M. Genes Cells. 2000; 5: 79-88Crossref PubMed Scopus (55) Google Scholar). If there is competition among populations of ς species for core binding, that competition may be influenced by binding specificity of the ςs. In light of the high sequence conservation of most ς species, it has been thought that all ς factors bind to the same location(s) on the core enzyme (1Helmann J.D. Chamberlin M.J. Annu. Rev. Biochem. 1988; 57: 839-872Crossref PubMed Scopus (716) Google Scholar). We had previously identified a binding site for ς70 in vitro (14Arthur T.M. Burgess R.R. J. Biol. Chem. 1998; 273: 31381-31387Abstract Full" @default.
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