Review
Mechanism of Bacterial Transcription Initiation: RNA Polymerase - Promoter Binding, Isomerization to Initiation-Competent Open Complexes, and Initiation of RNA Synthesis

https://doi.org/10.1016/j.jmb.2011.01.018Get rights and content

Abstract

Initiation of RNA synthesis from DNA templates by RNA polymerase (RNAP) is a multi-step process, in which initial recognition of promoter DNA by RNAP triggers a series of conformational changes in both RNAP and promoter DNA. The bacterial RNAP functions as a molecular isomerization machine, using binding free energy to remodel the initial recognition complex, placing downstream duplex DNA in the active site cleft and then separating the nontemplate and template strands in the region surrounding the start site of RNA synthesis. In this initial unstable “open” complex the template strand appears correctly positioned in the active site. Subsequently, the nontemplate strand is repositioned and a clamp is assembled on duplex DNA downstream of the open region to form the highly stable open complex, RPo. The transcription initiation factor, σ70, plays critical roles in promoter recognition and RPo formation as well as in early steps of RNA synthesis.

Introduction

We begin this perspective with a brief overview of transcription initiation by bacterial RNA polymerase (RNAP), summarizing the players and the major steps in the process. Excellent review articles provide a more detailed coverage of many aspects of transcription initiation.1, 2, 3, 4, 5, 6, 7, 8, 9 Here we focus on current advances in understanding the process of isomerization of the initial closed complex to form the stable open complex RPo and the many crucial roles of the specificity subunit σ70 in all steps of initiation.

Section snippets

The essential players

The bacterial RNAP “core enzyme” (E) consists of five subunits, ββ′α2ω (see Fig. 1). The core enzyme is capable of nonspecific DNA binding and initiation of RNA synthesis from DNA ends or nicks, but requires a sigma factor to initiate specific transcription from promoter DNA. Sigma assembles with core to form the “holoenzyme” (or Eσ).18, 19 Sigma factors recognize specific promoter DNA sequences, interact with transcription activators, participate in promoter DNA opening, and influence the

Mechanistic studies

How is the start site DNA opened, placed in the active site, and stabilized? During RPo formation, how and when are the obstacles that prevent nonpromoter DNA from accessing the cleft and being opened overcome? For several decades, kinetic mechanistic and footprinting studies have been employed to determine the sequence of conformational changes and the nature of intermediate complexes on the pathway from the initial promoter-recognition complex RPc to RPo. At the lac UV5 and λPR promoters, at

Promoter recognition

Structures of RNAP holoenzyme from Thermus aquaticus and Thermus thermophilus reveal that σ70 consists of several independently folded domains (σ1.2, σ2, σ4, and likely the N-terminal ∼ 60 residues of σ1.1 as well103) connected by flexible linkers (σ3.2 and the highly negatively charged C-terminal residues of σ1.1). Recent evidence reveals that the structure of the free sigma factors is compact, and that σ1.1 and σ4 interact.103 This interaction may lead to the observed autoinhibition of

Conclusion

Determination of high-resolution structures of free and promoter-bound holoenzymes, together with advances in our understanding of how salts and solutes interact with biopolymer surfaces and perturb biopolymer processes, has led to rapid progress in our understanding of the events of RNAP recruitment and promoter recognition to form the initial closed complex RPc, and the massive conformational changes in RNAP and promoter DNA that occur to convert it to the most stable open complex RPo.

Acknowledgements

We thank the reviewers for helpful comments, R. H. Ebright for conversations and communication of unpublished results, and our current and former colleagues for many fruitful collaborations. We thank Caroline Davis, Wayne Kontur, Theodore Gries, and Lisa Schroeder for their significant contributions to the mechanistic work reviewed here. We gratefully acknowledge Craig Bingman and Irina Artsimovitch for discussion of and assistance with Fig. 1, Fig. 3 and Supplementary Fig. 1, and Olga

References (140)

  • MooneyR.A. et al.

    Sigma and RNA polymerase: an on-again, off-again relationship?

    Mol. Cell

    (2005)
  • YoungB.A. et al.

    A coiled-coil from the RNA polymerase β subunit allosterically induces selective nontemplate strand binding by σ70

    Cell

    (2001)
  • RingB.Z. et al.

    Function of a nontranscribed DNA strand site in transcription elongation

    Cell

    (1994)
  • ZhangG. et al.

    Crystal structure of Thermus aquaticus core RNA polymerase at 3.3 Å resolution

    Cell

    (1999)
  • SaeckerR.M. et al.

    Kinetic studies and structural models of the association of E. coli σ70 RNA polymerase with the lambdaP(R) promoter: large scale conformational changes in forming the kinetically significant intermediates

    J. Mol. Biol.

    (2002)
  • MurakamiK.S. et al.

    Crystallographic analysis of Thermus aquaticus RNA polymerase holoenzyme and a holoenzyme/promoter DNA complex

    Methods Enzymol.

    (2003)
  • K.S. Murakami et al.

    Bacterial RNA polymerases: the wholo story

    Curr. Opin. Struct. Biol.

    (2003)
  • HoM.X. et al.

    Structures of RNA polymerase–antibiotic complexes

    Curr. Opin. Struct. Biol.

    (2009)
  • MeklerV. et al.

    Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase–promoter open complex

    Cell

    (2002)
  • RoeJ.H. et al.

    Kinetics and mechanism of the interaction of Escherichia coli RNA polymerase with the λPR promoter

    J. Mol. Biol.

    (1984)
  • RoeJ.H. et al.

    Temperature dependence of the rate constants of the Escherichia coli RNA polymerase–λPR promoter interaction. Assignment of the kinetic steps corresponding to protein conformational change and DNA opening

    J. Mol. Biol.

    (1985)
  • CraigM.L. et al.

    DNA footprints of the two kinetically significant intermediates in formation of an RNA polymerase–promoter open complex: evidence that interactions with start site and downstream DNA induce sequential conformational changes in polymerase and DNA

    J. Mol. Biol.

    (1998)
  • KovacicR.T.

    The 0 °C closed complexes between Escherichia coli RNA polymerase and two promoters, T7-A3 and lacUV5

    J. Biol. Chem.

    (1987)
  • CowingD.W. et al.

    Intermediates in the formation of the open complex by RNA polymerase holoenzyme containing the sigma factor σ32 at the groE promoter

    J. Mol. Biol.

    (1989)
  • SchroederL.A. et al.

    Evidence for a tyrosine–adenine stacking interaction and for a short-lived open intermediate subsequent to initial binding of Escherichia coli RNA polymerase to promoter DNA

    J. Mol. Biol.

    (2009)
  • TsodikovO.V. et al.

    Quantitative analysis of multiple-hit footprinting studies to characterize DNA conformational changes in protein–DNA complexes: application to DNA opening by Eσ70 RNA polymerase

    J. Mol. Biol.

    (1998)
  • K.S. Murakami et al.

    Crystallographic analysis of Thermus aquaticus RNA polymerase holoenzymeand a holoenzyme/promoter DNA complex

    Methods Enzymol.

    (2003)
  • MecsasJ. et al.

    Development of RNA polymerase–promoter contacts during open complex formation

    J. Mol. Biol.

    (1991)
  • BartlettM.S. et al.

    RNA polymerase mutants that destabilize RNA polymerase–promoter complexes alter NTP-sensing by rrn P1 promoters

    J. Mol. Biol.

    (1998)
  • LiX.Y. et al.

    Characterization of the closed complex intermediate formed during transcription initiation by Escherichia coli RNA polymerase

    J. Biol. Chem.

    (1998)
  • BusbyS. et al.

    Transcription activation by catabolite activator protein (CAP)

    J. Mol. Biol.

    (1999)
  • GiladiH. et al.

    Stimulation of the phage lambda pL promoter by integration host factor requires the carboxy terminus of the α-subunit of RNA polymerase

    J. Mol. Biol.

    (1992)
  • ChenH. et al.

    Functional interaction between RNA polymerase α subunit C-terminal domain and σ70 in UP-element- and activator-dependent transcription

    Mol. Cell

    (2003)
  • LeeD.J. et al.

    Exploitation of a chemical nuclease to investigate the location and orientation of the Escherichia coli RNA polymerase α subunit C-terminal domains at simple promoters that are activated by cyclic AMP receptor protein

    J. Biol. Chem.

    (2003)
  • LonettoM.A. et al.

    Identification of a contact site for different transcription activators in region 4 of the Escherichia coli RNA polymerase σ70 subunit

    J. Mol. Biol.

    (1998)
  • EderthJ. et al.

    The downstream DNA jaw of bacterial RNA polymerase facilitates both transcriptional initiation and pausing

    J. Biol. Chem.

    (2002)
  • BrowningD.F. et al.

    The regulation of bacterial transcription initiation

    Nat. Rev. Microbiol.

    (2004)
  • HaugenS.P. et al.

    Advances in bacterial promoter recognition and its control by factors that do not bind DNA

    Nat. Rev. Microbiol.

    (2008)
  • WigneshwerarajS. et al.

    Modus operandi of the bacterial RNA polymerase containing the σ54 promoter-specificity factor

    Mol. Microbiol.

    (2008)
  • deHasethP.L. et al.

    RNA polymerase–promoter interactions: the comings and goings of RNA polymerase

    J. Bacteriol.

    (1998)
  • GruberT.M. et al.

    Multiple sigma subunits and the partitioning of bacterial transcription space

    Annu. Rev. Microbiol.

    (2003)
  • PagetM.S. et al.

    The σ70 family of sigma factors

    Genome Biol.

    (2003)
  • Hook-BarnardI.G. et al.

    Transcription initiation by mix and match elements: flexibility for polymerase binding to bacterial promoters

    Gene Regul. Syst. Biol.

    (2007)
  • GhoshT. et al.

    Mechanisms for activating bacterial RNA polymerase

    FEMS Microbiol. Rev.

    (2010)
  • HudsonB.P. et al.

    Three-dimensional EM structure of an intact activator-dependent transcription initiation complex

    Proc. Natl Acad. Sci. USA

    (2009)
  • OpalkaN. et al.

    Complete structural model of Escherichia coli RNA polymerase from a hybrid approach

    PLoS Biol.

    (2010)
  • KonturW.S. et al.

    Solute probes of conformational changes in open complex (RPo) formation by Escherichia coli RNA polymerase at the λPR promoter: evidence for unmasking of the active site in the isomerization step and for large-scale coupled folding in the subsequent conversion to RPo

    Biochemistry

    (2006)
  • W.S. Kontur et al.

    Probing DNA binding, DNA opening and assembly of downstream clamp/jaw in Escherichia coli RNA polymerase–λPR promoter complexes using salt and the physiological anion glutamate

    Biochemistry

    (2010)
  • DavisC.A. et al.

    Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase

    Proc. Natl Acad. Sci. USA

    (2007)
  • VassylyevD.G. et al.

    Structural basis for transcription elongation by bacterial RNA polymerase

    Nature

    (2007)
  • Cited by (248)

    • Prokaryotic Transcription

      2022, Encyclopedia of Cell Biology: Volume 1-6, Second Edition
    View all citing articles on Scopus
    View full text