title: TFE and Spt4/5 open and close the RNA polymerase clamp during the transcription cycle.
abstract: Transcription is an intrinsically dynamic process and requires the coordinated interplay of RNA polymerases (RNAPs) with nucleic acids and transcription factors. Classical structural biology techniques have revealed detailed snapshots of a subset of conformational states of the RNAP as they exist in crystals. A detailed view of the conformational space sampled by the RNAP and the molecular mechanisms of the basal transcription factors E (TFE) and Spt4/5 through conformational constraints has remained elusive. We monitored the conformational changes of the flexible clamp of the RNAP by combining a fluorescently labeled recombinant 12-subunit RNAP system with single-molecule FRET measurements. We measured and compared the distances across the DNA binding channel of the archaeal RNAP. Our results show that the transition of the closed to the open initiation complex, which occurs concomitant with DNA melting, is coordinated with an opening of the RNAP clamp that is stimulated by TFE. We show that the clamp in elongation complexes is modulated by the nontemplate strand and by the processivity factor Spt4/5, both of which stimulate transcription processivity. Taken together, our results reveal an intricate network of interactions within transcription complexes between RNAP, transcription factors, and nucleic acids that allosterically modulate the RNAP during the transcription cycle.
authors.affiliation: Physikalische und Theoretische Chemie-NanoBioSciences, Technische Universität Braunschweig, 38106 Braunschweig, Germany;
authors.forename: Sarah
authors.initials: S
authors.lastname: Schulz
authors.affiliation: Physikalische und Theoretische Chemie-NanoBioSciences, Technische Universität Braunschweig, 38106 Braunschweig, Germany;
authors.forename: Andreas
authors.initials: A
authors.lastname: Gietl
authors.affiliation: RNA Polymerase Laboratory, Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London WC1E 6BT, United Kingdom;
authors.forename: Katherine
authors.initials: K
authors.lastname: Smollett
authors.affiliation: Physikalische und Theoretische Chemie-NanoBioSciences, Technische Universität Braunschweig, 38106 Braunschweig, Germany; Braunschweig Integrated Centre of Systems Biology (BRICS), Technische Universität Braunschweig, 38106 Braunschweig, Germany; Laboratory for Emerging Nanometrology (LENA), Technische Universität Braunschweig, 38106 Braunschweig, Germany.
authors.forename: Philip
authors.initials: P
authors.lastname: Tinnefeld
authors.affiliation: RNA Polymerase Laboratory, Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London WC1E 6BT, United Kingdom; f.werner@ucl.ac.uk dina.grohmann@ur.de.
authors.forename: Finn
authors.initials: F
authors.lastname: Werner
authors.affiliation: Physikalische und Theoretische Chemie-NanoBioSciences, Technische Universität Braunschweig, 38106 Braunschweig, Germany; f.werner@ucl.ac.uk dina.grohmann@ur.de.
authors.forename: Dina
authors.initials: D
authors.lastname: Grohmann
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D018360:Crystallography, X-Ray; D019641:DNA, Archaeal / Q000737:chemistry / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D063747:Methanocaldococcus / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors / Q000737:chemistry / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D009711:Nucleotides; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D012321:DNA-Directed RNA Polymerases
keywords: RNA polymerase; archaea; clamp; single-molecule FRET; transcription