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• W1970377220 abstract "Transcription, the synthesis of RNA from a DNA template, is performed by multisubunit RNA polymerases (RNAPs) in all cellular organisms. The bridge helix (BH) is a distinct feature of all multisubunit RNAPs and makes direct interactions with several active site-associated mobile features implicated in the nucleotide addition cycle and RNA and DNA binding. Because the BH has been captured in both kinked and straight conformations in different crystals structures of RNAP, recently supported by molecular dynamics studies, it has been proposed that cycling between these conformations is an integral part of the nucleotide addition cycle. To further evaluate the role of the BH, we conducted systematic alanine scanning mutagenesis of the Escherichia coli RNAP BH to determine its contributions to activities required for transcription. Combining our data with an atomic model of E. coli RNAP, we suggest that alterations in the interactions between the BH and (i) the trigger loop, (ii) fork loop 2, and (iii) switch 2 can help explain the observed changes in RNAP functionality associated with some of the BH variants. Additionally, we show that extensive defects in E. coli RNAP functionality depend upon a single previously not studied lysine residue (Lys-781) that is strictly conserved in all bacteria. It appears that direct interactions made by the BH with other conserved features of RNAP are lost in some of the E. coli alanine substitution variants, which we infer results in conformational changes in RNAP that modify RNAP functionality. Transcription, the synthesis of RNA from a DNA template, is performed by multisubunit RNA polymerases (RNAPs) in all cellular organisms. The bridge helix (BH) is a distinct feature of all multisubunit RNAPs and makes direct interactions with several active site-associated mobile features implicated in the nucleotide addition cycle and RNA and DNA binding. Because the BH has been captured in both kinked and straight conformations in different crystals structures of RNAP, recently supported by molecular dynamics studies, it has been proposed that cycling between these conformations is an integral part of the nucleotide addition cycle. To further evaluate the role of the BH, we conducted systematic alanine scanning mutagenesis of the Escherichia coli RNAP BH to determine its contributions to activities required for transcription. Combining our data with an atomic model of E. coli RNAP, we suggest that alterations in the interactions between the BH and (i) the trigger loop, (ii) fork loop 2, and (iii) switch 2 can help explain the observed changes in RNAP functionality associated with some of the BH variants. Additionally, we show that extensive defects in E. coli RNAP functionality depend upon a single previously not studied lysine residue (Lys-781) that is strictly conserved in all bacteria. It appears that direct interactions made by the BH with other conserved features of RNAP are lost in some of the E. coli alanine substitution variants, which we infer results in conformational changes in RNAP that modify RNAP functionality. IntroductionMultisubunit RNA polymerases (RNAPs) 3The abbreviations used are: RNAP, RNA polymerase; BH, bridge helix; FL2, fork loop 2; TL, trigger loop; STA, srandard transcription assay. function as complex molecular machines that are central to regulated gene expression in all cellular organisms (1Cramer P. Arnold E. Curr. Opin. Struct. Biol. 2009; 19: 680-682Crossref PubMed Scopus (5) Google Scholar, 2Lane W.J. Darst S.A. J. Mol. Biol. 2010; 395: 671-685Crossref PubMed Scopus (120) Google Scholar, 3Lane W.J. Darst S.A. J. Mol. Biol. 2010; 395: 686-704Crossref PubMed Scopus (94) Google Scholar). Multiple conformational changes accompany and are required for the action of RNAP. These can be modulated in magnitude and frequency by control factors that act as regulatory signals. The catalytically competent core enzyme (α2ββ′ω; in bacteria) is highly conserved across all domains of life in its ternary structure and sequence (1Cramer P. Arnold E. Curr. Opin. Struct. Biol. 2009; 19: 680-682Crossref PubMed Scopus (5) Google Scholar, 2Lane W.J. Darst S.A. J. Mol. Biol. 2010; 395: 671-685Crossref PubMed Scopus (120) Google Scholar, 3Lane W.J. Darst S.A. J. Mol. Biol. 2010; 395: 686-704Crossref PubMed Scopus (94) Google Scholar, 4Werner F. Trends Microbiol. 2008; 16: 247-250Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The acquisition of high resolution structures of RNAPs and their complexes from bacteria, yeast, and archaea, coupled with biochemical and biophysical analyses of RNAP domain functionalities, has led to a detailed molecular level appreciation of how the enzyme functions in promoter recognition, transcription initiation, elongation, and pausing (1Cramer P. Arnold E. Curr. Opin. Struct. Biol. 2009; 19: 680-682Crossref PubMed Scopus (5) Google Scholar, 3Lane W.J. Darst S.A. J. Mol. Biol. 2010; 395: 686-704Crossref PubMed Scopus (94) Google Scholar, 5Nudler E. Annu. Rev. Biochem. 2009; 78: 335-361Crossref PubMed Scopus (109) Google Scholar).One prominent conserved structural feature of RNAP is the β′ bridge helix (BH) (see Fig. 1A), an α-helical domain that spans the active site of the enzyme (5Nudler E. Annu. Rev. Biochem. 2009; 78: 335-361Crossref PubMed Scopus (109) Google Scholar, 6Svetlov V. Nudler E. Curr. Opin. Struct. Biol. 2009; 19: 701-707Crossref PubMed Scopus (35) Google Scholar). Other conserved structural features of the RNAP active center include β fork loop 2 (FL2), which maintains the downstream edge of the transcription bubble, and the β′ lid domain, which helps maintain the upstream edge of the RNA-DNA hybrid (3Lane W.J. Darst S.A. J. Mol. Biol. 2010; 395: 686-704Crossref PubMed Scopus (94) Google Scholar, 5Nudler E. Annu. Rev. Biochem. 2009; 78: 335-361Crossref PubMed Scopus (109) Google Scholar, 7Korzheva N. Mustaev A. Kozlov M. Malhotra A. Nikiforov V. Goldfarb A. Darst S.A. Science. 2000; 289: 619-625Crossref PubMed Scopus (338) Google Scholar, 8Vassylyev D.G. Vassylyeva M.N. Perederina A. Tahirov T.H. Artsimovitch I. Nature. 2007; 448: 157-162Crossref PubMed Scopus (326) Google Scholar). Along with the BH, another feature of the RNAP catalytic center is the trigger loop (TL); both are involved in NTP binding and translocation and have been captured in distinctly different conformations, implying that they possess dynamic characteristics (9Cramer P. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (955) Google Scholar, 10Gnatt A.L. Cramer P. Fu J. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1876-1882Crossref PubMed Scopus (742) Google Scholar, 11Tuske S. Sarafianos S.G. Wang X. Hudson B. Sineva E. Mukhopadhyay J. Birktoft J.J. Leroy O. Ismail S. Clark Jr., A.D. Dharia C. Napoli A. Laptenko O. Lee J. Borukhov S. Ebright R.H. Arnold E. Cell. 2005; 122: 541-552Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 12Vassylyev D.G. Sekine S. Laptenko O. Lee J. Vassylyeva M.N. Borukhov S. Yokoyama S. Nature. 2002; 417: 712-719Crossref PubMed Scopus (623) Google Scholar, 13Vassylyev D.G. Vassylyeva M.N. Zhang J. Palangat M. Artsimovitch I. Landick R. Nature. 2007; 448: 163-168Crossref PubMed Scopus (283) Google Scholar, 14Wang D. Bushnell D.A. Westover K.D. Kaplan C.D. Kornberg R.D. Cell. 2006; 127: 941-954Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 15Zhang 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 (666) Google Scholar). Proposed mechanisms of the translocation event, associated with extension of the RNA chain, postulate that binding of the incoming NTP is coupled with a conformational change in the BH-TL assemblage for stepwise forward movement of RNAP (7Korzheva N. Mustaev A. Kozlov M. Malhotra A. Nikiforov V. Goldfarb A. Darst S.A. Science. 2000; 289: 619-625Crossref PubMed Scopus (338) Google Scholar, 9Cramer P. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (955) Google Scholar, 13Vassylyev D.G. Vassylyeva M.N. Zhang J. Palangat M. Artsimovitch I. Landick R. Nature. 2007; 448: 163-168Crossref PubMed Scopus (283) Google Scholar, 14Wang D. Bushnell D.A. Westover K.D. Kaplan C.D. Kornberg R.D. Cell. 2006; 127: 941-954Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 16Brueckner F. Cramer P. Nat. Struct. Mol. Biol. 2008; 15: 811-818Crossref PubMed Scopus (185) Google Scholar, 17Cramer P. Curr. Opin. Struct. Biol. 2002; 12: 89-97Crossref PubMed Scopus (187) Google Scholar). An additional feature of the RNAP catalytic site, the β′ F-loop (also termed conserved region β′a15) (3Lane W.J. Darst S.A. J. Mol. Biol. 2010; 395: 686-704Crossref PubMed Scopus (94) Google Scholar), has also been demonstrated to play an essential role (alongside the BH and TL) in catalysis by bacterial RNAPs (18Miropolskaya N. Artsimovitch I. Klimasauskas S. Nikiforov V. Kulbachinskiy A. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 18942-18947Crossref PubMed Scopus (36) Google Scholar). Several RNAP-interacting factors function through or with the BH and TL. The phytotoxin tagetitoxin binds in the vicinity of the active center, inhibiting RNAP elongation (19Vassylyev D.G. Svetlov V. Vassylyeva M.N. Perederina A. Igarashi N. Matsugaki N. Wakatsuki S. Artsimovitch I. Nat. Struct. Mol. Biol. 2005; 12: 1086-1093Crossref PubMed Scopus (65) Google Scholar) potentially by restricting movements of the BH and TL. Streptolydigin binds to the BH and interferes with the nucleotide addition cycle, thereby inhibiting RNAP activity (11Tuske S. Sarafianos S.G. Wang X. Hudson B. Sineva E. Mukhopadhyay J. Birktoft J.J. Leroy O. Ismail S. Clark Jr., A.D. Dharia C. Napoli A. Laptenko O. Lee J. Borukhov S. Ebright R.H. Arnold E. Cell. 2005; 122: 541-552Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 20Temiakov D. Zenkin N. Vassylyeva M.N. Perederina A. Tahirov T.H. Kashkina E. Savkina M. Zorov S. Nikiforov V. Igarashi N. Matsugaki N. Wakatsuki S. Severinov K. Vassylyev D.G. Mol. Cell. 2005; 19: 655-666Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Furthermore, the transcript cleavage factor Gre appears to assist in RNAP backtracking and stimulates RNA cleavage via the BH and TL (21Artsimovitch I. Chu C. Lynch A.S. Landick R. Science. 2003; 302: 650-654Crossref PubMed Scopus (85) Google Scholar, 22Borukhov S. Polyakov A. Nikiforov V. Goldfarb A. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 8899-8902Crossref PubMed Scopus (133) Google Scholar, 23Borukhov S. Sagitov V. Goldfarb A. Cell. 1993; 72: 459-466Abstract Full Text PDF PubMed Scopus (307) Google Scholar, 24Nickels B.E. Hochschild A. Cell. 2004; 118: 281-284Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar).Mutating residues in the BH can result in fast (F773V in Escherichia coli) (25Svetlov V. Belogurov G.A. Shabrova E. Vassylyev D.G. Artsimovitch I. Nucleic Acids Res. 2007; 35: 5694-5705Crossref PubMed Scopus (56) Google Scholar) or “super-active” (S824P in Methanocaldococcus jannaschii) (26Tan L. Wiesler S. Trzaska D. Carney H.C. Weinzierl R.O. J. Biol. 2008; 7: 40Crossref PubMed Scopus (70) Google Scholar) variants of RNAP. To gain further insights into the contributions the BH makes to RNAP activities, we conducted comprehensive alanine scanning mutagenesis of the BH feature of E. coli RNAP (see Fig. 1B). Mutants were assayed for (i) promoter-specific (i.e. σ-dependent), (ii) nonspecific promoter-independent, and (iii) specific (RNA primer-dependent) promoter-independent transcription activities (see “Experimental Procedures” and Fig. 1C), as well as effects on in vivo growth. Taken together, our results generate a comprehensive activity map of the E. coli RNAP BH (see Fig. 2). A number of residues important for transcript formation were identified. Inspection of an atomic model of E. coli RNAP (Protein Data Bank code 3LU0) (27Opalka N. Brown J. Lane W.J. Twist K.A. Landick R. Asturias F.J. Darst S.A. PLoS Biol. 2010; 8: e1000483Crossref PubMed Scopus (96) Google Scholar) determined that altered interactions between the BH and (i) the TL, (ii) the F-loop, (iii) FL2, and/or (iv) switch 2 of RNAP may be linked to the observed defects (in the BH alanine substitution mutants). In particular, lysine at position 781, which is invariant in bacteria, appears to be essential in all of the in vitro activities tested. The BH variants that fail in elongation and pyrophosphate-mediated RNA cleavage harbor potential defects in the catalytic activity used for RNA synthesis. BH variants that display significantly reduced in vivo growth activities (in contrast to their observed in vitro activities) could display defects not directly tested by our in vitro assays, e.g. promoter pausing. Finally, two BH variants (V801A and D802A) exhibited severely impaired minimal scaffold-binding activities; we infer that these represent forms of RNAP in which the clamp domain may adopt a more closed conformation.FIGURE 2Summary of the activities of the BH variants. Upper, atomic model of the E. coli BH with positions of the residues mutated in this study indicated. Lower, summary table listing the activities of the BH variants obtained using the assays performed in this study. Red designates severe (<20% of WT activity) defects, pink indicates reduced activity (<50% of WT activity), and blue denotes impaired minimal scaffold binding. The gray lettering refers to residues not mutated in this study. Sites in the M. jannaschii (Mj) BH denoted as the N- and C-terminal hinge regions (BH-HN and BH-HC, respectively), required for helix stabilization or to be structurally labile, are also shown (26Tan L. Wiesler S. Trzaska D. Carney H.C. Weinzierl R.O. J. Biol. 2008; 7: 40Crossref PubMed Scopus (70) Google Scholar, 39Weinzierl R.O. BMC Biol. 2010; 8: 134Crossref PubMed Scopus (37) Google Scholar, 49Weinzierl R.O. Biochem. Soc. Trans. 2010; 38: 428-432Crossref PubMed Scopus (10) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) IntroductionMultisubunit RNA polymerases (RNAPs) 3The abbreviations used are: RNAP, RNA polymerase; BH, bridge helix; FL2, fork loop 2; TL, trigger loop; STA, srandard transcription assay. function as complex molecular machines that are central to regulated gene expression in all cellular organisms (1Cramer P. Arnold E. Curr. Opin. Struct. Biol. 2009; 19: 680-682Crossref PubMed Scopus (5) Google Scholar, 2Lane W.J. Darst S.A. J. Mol. Biol. 2010; 395: 671-685Crossref PubMed Scopus (120) Google Scholar, 3Lane W.J. Darst S.A. J. Mol. Biol. 2010; 395: 686-704Crossref PubMed Scopus (94) Google Scholar). Multiple conformational changes accompany and are required for the action of RNAP. These can be modulated in magnitude and frequency by control factors that act as regulatory signals. The catalytically competent core enzyme (α2ββ′ω; in bacteria) is highly conserved across all domains of life in its ternary structure and sequence (1Cramer P. Arnold E. Curr. Opin. Struct. Biol. 2009; 19: 680-682Crossref PubMed Scopus (5) Google Scholar, 2Lane W.J. Darst S.A. J. Mol. Biol. 2010; 395: 671-685Crossref PubMed Scopus (120) Google Scholar, 3Lane W.J. Darst S.A. J. Mol. Biol. 2010; 395: 686-704Crossref PubMed Scopus (94) Google Scholar, 4Werner F. Trends Microbiol. 2008; 16: 247-250Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The acquisition of high resolution structures of RNAPs and their complexes from bacteria, yeast, and archaea, coupled with biochemical and biophysical analyses of RNAP domain functionalities, has led to a detailed molecular level appreciation of how the enzyme functions in promoter recognition, transcription initiation, elongation, and pausing (1Cramer P. Arnold E. Curr. Opin. Struct. Biol. 2009; 19: 680-682Crossref PubMed Scopus (5) Google Scholar, 3Lane W.J. Darst S.A. J. Mol. Biol. 2010; 395: 686-704Crossref PubMed Scopus (94) Google Scholar, 5Nudler E. Annu. Rev. Biochem. 2009; 78: 335-361Crossref PubMed Scopus (109) Google Scholar).One prominent conserved structural feature of RNAP is the β′ bridge helix (BH) (see Fig. 1A), an α-helical domain that spans the active site of the enzyme (5Nudler E. Annu. Rev. Biochem. 2009; 78: 335-361Crossref PubMed Scopus (109) Google Scholar, 6Svetlov V. Nudler E. Curr. Opin. Struct. Biol. 2009; 19: 701-707Crossref PubMed Scopus (35) Google Scholar). Other conserved structural features of the RNAP active center include β fork loop 2 (FL2), which maintains the downstream edge of the transcription bubble, and the β′ lid domain, which helps maintain the upstream edge of the RNA-DNA hybrid (3Lane W.J. Darst S.A. J. Mol. Biol. 2010; 395: 686-704Crossref PubMed Scopus (94) Google Scholar, 5Nudler E. Annu. Rev. Biochem. 2009; 78: 335-361Crossref PubMed Scopus (109) Google Scholar, 7Korzheva N. Mustaev A. Kozlov M. Malhotra A. Nikiforov V. Goldfarb A. Darst S.A. Science. 2000; 289: 619-625Crossref PubMed Scopus (338) Google Scholar, 8Vassylyev D.G. Vassylyeva M.N. Perederina A. Tahirov T.H. Artsimovitch I. Nature. 2007; 448: 157-162Crossref PubMed Scopus (326) Google Scholar). Along with the BH, another feature of the RNAP catalytic center is the trigger loop (TL); both are involved in NTP binding and translocation and have been captured in distinctly different conformations, implying that they possess dynamic characteristics (9Cramer P. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (955) Google Scholar, 10Gnatt A.L. Cramer P. Fu J. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1876-1882Crossref PubMed Scopus (742) Google Scholar, 11Tuske S. Sarafianos S.G. Wang X. Hudson B. Sineva E. Mukhopadhyay J. Birktoft J.J. Leroy O. Ismail S. Clark Jr., A.D. Dharia C. Napoli A. Laptenko O. Lee J. Borukhov S. Ebright R.H. Arnold E. Cell. 2005; 122: 541-552Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 12Vassylyev D.G. Sekine S. Laptenko O. Lee J. Vassylyeva M.N. Borukhov S. Yokoyama S. Nature. 2002; 417: 712-719Crossref PubMed Scopus (623) Google Scholar, 13Vassylyev D.G. Vassylyeva M.N. Zhang J. Palangat M. Artsimovitch I. Landick R. Nature. 2007; 448: 163-168Crossref PubMed Scopus (283) Google Scholar, 14Wang D. Bushnell D.A. Westover K.D. Kaplan C.D. Kornberg R.D. Cell. 2006; 127: 941-954Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 15Zhang 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 (666) Google Scholar). Proposed mechanisms of the translocation event, associated with extension of the RNA chain, postulate that binding of the incoming NTP is coupled with a conformational change in the BH-TL assemblage for stepwise forward movement of RNAP (7Korzheva N. Mustaev A. Kozlov M. Malhotra A. Nikiforov V. Goldfarb A. Darst S.A. Science. 2000; 289: 619-625Crossref PubMed Scopus (338) Google Scholar, 9Cramer P. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (955) Google Scholar, 13Vassylyev D.G. Vassylyeva M.N. Zhang J. Palangat M. Artsimovitch I. Landick R. Nature. 2007; 448: 163-168Crossref PubMed Scopus (283) Google Scholar, 14Wang D. Bushnell D.A. Westover K.D. Kaplan C.D. Kornberg R.D. Cell. 2006; 127: 941-954Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 16Brueckner F. Cramer P. Nat. Struct. Mol. Biol. 2008; 15: 811-818Crossref PubMed Scopus (185) Google Scholar, 17Cramer P. Curr. Opin. Struct. Biol. 2002; 12: 89-97Crossref PubMed Scopus (187) Google Scholar). An additional feature of the RNAP catalytic site, the β′ F-loop (also termed conserved region β′a15) (3Lane W.J. Darst S.A. J. Mol. Biol. 2010; 395: 686-704Crossref PubMed Scopus (94) Google Scholar), has also been demonstrated to play an essential role (alongside the BH and TL) in catalysis by bacterial RNAPs (18Miropolskaya N. Artsimovitch I. Klimasauskas S. Nikiforov V. Kulbachinskiy A. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 18942-18947Crossref PubMed Scopus (36) Google Scholar). Several RNAP-interacting factors function through or with the BH and TL. The phytotoxin tagetitoxin binds in the vicinity of the active center, inhibiting RNAP elongation (19Vassylyev D.G. Svetlov V. Vassylyeva M.N. Perederina A. Igarashi N. Matsugaki N. Wakatsuki S. Artsimovitch I. Nat. Struct. Mol. Biol. 2005; 12: 1086-1093Crossref PubMed Scopus (65) Google Scholar) potentially by restricting movements of the BH and TL. Streptolydigin binds to the BH and interferes with the nucleotide addition cycle, thereby inhibiting RNAP activity (11Tuske S. Sarafianos S.G. Wang X. Hudson B. Sineva E. Mukhopadhyay J. Birktoft J.J. Leroy O. Ismail S. Clark Jr., A.D. Dharia C. Napoli A. Laptenko O. Lee J. Borukhov S. Ebright R.H. Arnold E. Cell. 2005; 122: 541-552Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 20Temiakov D. Zenkin N. Vassylyeva M.N. Perederina A. Tahirov T.H. Kashkina E. Savkina M. Zorov S. Nikiforov V. Igarashi N. Matsugaki N. Wakatsuki S. Severinov K. Vassylyev D.G. Mol. Cell. 2005; 19: 655-666Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Furthermore, the transcript cleavage factor Gre appears to assist in RNAP backtracking and stimulates RNA cleavage via the BH and TL (21Artsimovitch I. Chu C. Lynch A.S. Landick R. Science. 2003; 302: 650-654Crossref PubMed Scopus (85) Google Scholar, 22Borukhov S. Polyakov A. Nikiforov V. Goldfarb A. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 8899-8902Crossref PubMed Scopus (133) Google Scholar, 23Borukhov S. Sagitov V. Goldfarb A. Cell. 1993; 72: 459-466Abstract Full Text PDF PubMed Scopus (307) Google Scholar, 24Nickels B.E. Hochschild A. Cell. 2004; 118: 281-284Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar).Mutating residues in the BH can result in fast (F773V in Escherichia coli) (25Svetlov V. Belogurov G.A. Shabrova E. Vassylyev D.G. Artsimovitch I. Nucleic Acids Res. 2007; 35: 5694-5705Crossref PubMed Scopus (56) Google Scholar) or “super-active” (S824P in Methanocaldococcus jannaschii) (26Tan L. Wiesler S. Trzaska D. Carney H.C. Weinzierl R.O. J. Biol. 2008; 7: 40Crossref PubMed Scopus (70) Google Scholar) variants of RNAP. To gain further insights into the contributions the BH makes to RNAP activities, we conducted comprehensive alanine scanning mutagenesis of the BH feature of E. coli RNAP (see Fig. 1B). Mutants were assayed for (i) promoter-specific (i.e. σ-dependent), (ii) nonspecific promoter-independent, and (iii) specific (RNA primer-dependent) promoter-independent transcription activities (see “Experimental Procedures” and Fig. 1C), as well as effects on in vivo growth. Taken together, our results generate a comprehensive activity map of the E. coli RNAP BH (see Fig. 2). A number of residues important for transcript formation were identified. Inspection of an atomic model of E. coli RNAP (Protein Data Bank code 3LU0) (27Opalka N. Brown J. Lane W.J. Twist K.A. Landick R. Asturias F.J. Darst S.A. PLoS Biol. 2010; 8: e1000483Crossref PubMed Scopus (96) Google Scholar) determined that altered interactions between the BH and (i) the TL, (ii) the F-loop, (iii) FL2, and/or (iv) switch 2 of RNAP may be linked to the observed defects (in the BH alanine substitution mutants). In particular, lysine at position 781, which is invariant in bacteria, appears to be essential in all of the in vitro activities tested. The BH variants that fail in elongation and pyrophosphate-mediated RNA cleavage harbor potential defects in the catalytic activity used for RNA synthesis. BH variants that display significantly reduced in vivo growth activities (in contrast to their observed in vitro activities) could display defects not directly tested by our in vitro assays, e.g. promoter pausing. Finally, two BH variants (V801A and D802A) exhibited severely impaired minimal scaffold-binding activities; we infer that these represent forms of RNAP in which the clamp domain may adopt a more closed conformation." @default.
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• W1970377220 date "2011-04-01" @default.
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• W1970377220 title "Activity Map of the Escherichia coli RNA Polymerase Bridge Helix" @default.
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