title: Mutations in RNA Polymerase Bridge Helix and Switch Regions Affect Active-Site Networks and Transcript-Assisted Hydrolysis.
abstract: In bacterial RNA polymerase (RNAP), the bridge helix and switch regions form an intricate network with the catalytic active centre and the main channel. These interactions are important for catalysis, hydrolysis and clamp domain movement. By targeting conserved residues in Escherichia coli RNAP, we are able to show that functions of these regions are differentially required during σ(70)-dependent and the contrasting σ(54)-dependent transcription activations and thus potentially underlie the key mechanistic differences between the two transcription paradigms. We further demonstrate that the transcription factor DksA directly regulates σ(54)-dependent activation both positively and negatively. This finding is consistent with the observed impacts of DksA on σ(70)-dependent promoters. DksA does not seem to significantly affect RNAP binding to a pre-melted promoter DNA but affects extensively activity at the stage of initial RNA synthesis on σ(54)-regulated promoters. Strikingly, removal of the σ(54) Region I is sufficient to invert the action of DksA (from stimulation to inhibition or vice versa) at two test promoters. The RNAP mutants we generated also show a strong propensity to backtrack. These mutants increase the rate of transcript-hydrolysis cleavage to a level comparable to that seen in the Thermus aquaticus RNAP even in the absence of a non-complementary nucleotide. These novel phenotypes imply an important function of the bridge helix and switch regions as an anti-backtracking ratchet and an RNA hydrolysis regulator.
authors.affiliation: Division of Cell and Molecular Biology, Imperial College London, Sir Alexander Fleming Building, Exhibition Road, London SW7 2AZ, United Kingdom. Electronic address: nan.zhang@imperial.ac.uk.
authors.forename: Nan
authors.initials: N
authors.lastname: Zhang
authors.affiliation: Division of Cell and Molecular Biology, Imperial College London, Sir Alexander Fleming Building, Exhibition Road, London SW7 2AZ, United Kingdom.
authors.forename: Jorrit
authors.initials: J
authors.lastname: Schäfer
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Amit
authors.initials: A
authors.lastname: Sharma
authors.affiliation: Division of Cell and Molecular Biology, Imperial College London, Sir Alexander Fleming Building, Exhibition Road, London SW7 2AZ, United Kingdom.
authors.forename: Lucy
authors.initials: L
authors.lastname: Rayner
authors.affiliation: Division of Macromolecular Structure and Function, Imperial College London, Exhibition Road, London SW7 2AZ, United Kingdom.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Roman
authors.initials: R
authors.lastname: Tuma
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Peter
authors.initials: P
authors.lastname: Stockley
authors.affiliation: Division of Cell and Molecular Biology, Imperial College London, Sir Alexander Fleming Building, Exhibition Road, London SW7 2AZ, United Kingdom. Electronic address: m.buck@imperial.ac.uk.
authors.forename: Martin
authors.initials: M
authors.lastname: Buck
mesh_terms: D020134:Catalytic Domain; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006868:Hydrolysis; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D051877:RNA Polymerase Sigma 54 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014158:Transcription, Genetic / Q000235:genetics*
chemical_list: D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012808:Sigma Factor; C090751:dksA protein, E coli; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54
keywords: DksA; RNA hydrolysis; RNA polymerase; bridge helix; switch regions