title: Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase.
abstract: Initiation is a highly regulated, rate-limiting step in transcription. We used a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with gel-based assays, showed that RNAP exit kinetics from complexes stalled at later stages of initiation (e.g., from a 7-base transcript) were markedly slower than from earlier stages (e.g., from a 2- or 4-base transcript). In addition, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states. Further examination with magnetic tweezers transcription experiments showed that RNAP adopted a long-lived backtracked state during initiation and that the paused-backtracked initiation intermediate was populated abundantly at physiologically relevant nucleoside triphosphate (NTP) concentrations. The paused intermediate population was further increased when the NTP concentration was decreased and/or when an imbalance in NTP concentration was introduced (situations that mimic stress). Our results confirm the existence of a previously hypothesized paused and backtracked RNAP initiation intermediate and suggest it is biologically relevant; furthermore, such intermediates could be exploited for therapeutic purposes and may reflect a conserved state among paused, initiating eukaryotic RNA polymerase II enzymes.
authors.affiliation: Department of Chemistry & Biochemistry, University of California, Los Angeles, CA 90095.
authors.forename: Eitan
authors.initials: E
authors.lastname: Lerner
authors.affiliation: Department of Chemistry & Biochemistry, University of California, Los Angeles, CA 90095.
authors.forename: SangYoon
authors.initials: S
authors.lastname: Chung
authors.affiliation: Department of Chemistry & Biochemistry, University of Colorado, Boulder, CO 80309.
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Allen
authors.affiliation: Institut Jacques Monod, Centre National de la Recherche Scientifique (CNRS), UMR7592, University Paris Diderot, Sorbonne Paris Cité, F-75205 Paris, France.
authors.forename: Shuang
authors.initials: S
authors.lastname: Wang
authors.affiliation: Rowan University School of Osteopathic Medicine, Stratford, NJ 08084.
authors.forename: Jookyung
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Chemistry & Biochemistry, University of California, Los Angeles, CA 90095.
authors.forename: Shijia W
authors.initials: SW
authors.lastname: Lu
authors.affiliation: Department of Chemistry & Biochemistry, University of California, Los Angeles, CA 90095.
authors.forename: Logan W
authors.initials: LW
authors.lastname: Grimaud
authors.affiliation: Department of Chemistry & Biochemistry, University of California, Los Angeles, CA 90095.
authors.forename: Antonino
authors.identifier: 0000-0002-9348-1397
authors.initials: A
authors.lastname: Ingargiola
authors.affiliation: Department of Chemistry & Biochemistry, University of California, Los Angeles, CA 90095.
authors.forename: Xavier
authors.initials: X
authors.lastname: Michalet
authors.affiliation: Department of Chemistry & Biochemistry, University of California, Los Angeles, CA 90095.
authors.forename: Yazan
authors.initials: Y
authors.lastname: Alhadid
authors.affiliation: Rowan University School of Osteopathic Medicine, Stratford, NJ 08084.
authors.forename: Sergei
authors.initials: S
authors.lastname: Borukhov
authors.affiliation: Institut Jacques Monod, Centre National de la Recherche Scientifique (CNRS), UMR7592, University Paris Diderot, Sorbonne Paris Cité, F-75205 Paris, France; Institut de Biologie de l'Ecole Normale Supérieure, Institut de Biologie de l'Ecole Normale Superieure (IBENS), CNRS, Inserm, Ecole Normale Supérieure, Paris Sciences et Lettres (PSL) Research University, F-75005 Paris, France; Programme Equipe Labellisées, Ligue Contre le Cancer, 75013 Paris, France; sweiss@chem.ucla.edu strick@biologie.ens.fr taatjes@colorado.edu.
authors.forename: Terence R
authors.initials: TR
authors.lastname: Strick
authors.affiliation: Department of Chemistry & Biochemistry, University of Colorado, Boulder, CO 80309; sweiss@chem.ucla.edu strick@biologie.ens.fr taatjes@colorado.edu.
authors.forename: Dylan J
authors.initials: DJ
authors.lastname: Taatjes
authors.affiliation: Department of Chemistry & Biochemistry, University of California, Los Angeles, CA 90095; Molecular Biology Institute, University of California, Los Angeles, CA 90095; Department of Physiology, University of California, Los Angeles, CA 90095 sweiss@chem.ucla.edu strick@biologie.ens.fr taatjes@colorado.edu.
authors.forename: Shimon
authors.initials: S
authors.lastname: Weiss
mesh_terms: D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D009690:Nucleic Acid Conformation; D012319:RNA Polymerase II / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D061785:Transcription Initiation, Genetic; D014544:Uridine Triphosphate / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; C079668:GreA protein, E coli; D012333:RNA, Messenger; D014157:Transcription Factors; D006160:Guanosine Triphosphate; D012319:RNA Polymerase II; D014544:Uridine Triphosphate
keywords: RNA polymerase; RNAP; backtracking; pausing; transcription