Structures of Bacterial RNA Polymerase Complexes Reveal the Mechanism of DNA Loading and Transcription Initiation

Summary Gene transcription is carried out by multi-subunit RNA polymerases (RNAPs). Transcription initiation is a dynamic multi-step process that involves the opening of the double-stranded DNA to form a transcription bubble and delivery of the template strand deep into the RNAP for RNA synthesis. Applying cryoelectron microscopy to a unique transcription system using σ54 (σN), the major bacterial variant sigma factor, we capture a new intermediate state at 4.1 Å where promoter DNA is caught at the entrance of the RNAP cleft. Combining with new structures of the open promoter complex and an initial de novo transcribing complex at 3.4 and 3.7 Å, respectively, our studies reveal the dynamics of DNA loading and mechanism of transcription bubble stabilization that involves coordinated, large-scale conformational changes of the universally conserved features within RNAP and DNA. In addition, our studies reveal a novel mechanism of strand separation by σ54.

• Publication year

  2018

• Venue

  Molecules and Cells

• Publication date

  2018-06-01

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1016/j.molcel.2018.05.021PMID 29932903PMCID 6028918
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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• Structural basis of RNA polymerase III transcription initiation

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• Molecular mechanism of promoter opening by RNA polymerase III

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  2017influential reference
• Structures of transcription pre-initiation complex with TFIIH and Mediator

  2017cited by this paper
• RNA polymerase motions during promoter melting

  2017influential reference
• Structural Insights into the Eukaryotic Transcription Initiation Machinery.

  2017cited by this paper
• Transcription initiation complex structures elucidate DNA opening

  2016influential reference
• Molecular Mechanisms of Transcription Initiation-Structure, Function, and Evolution of TFE/TFIIE-Like Factors and Open Complex Formation.

  2016cited by this paper
• Near-atomic resolution visualization of human transcription promoter opening

  2016influential reference
• The Structural Basis of Transcription: 10 Years After the Nobel Prize in Chemistry.

  2016cited by this paper
• Local and global regulation of transcription initiation in bacteria

  2016cited by this paper
• Gctf: Real-time CTF determination and correction

  2015cited by this paper
• Structural Biology of Bacterial RNA Polymerase

  2015cited by this paper
• Crystal structures of the E. coli transcription initiation complexes with a complete bubble.

  2015influential reference
• Structure of an RNA polymerase II preinitiation complex

  2015cited by this paper
• Author response: Structure of a bacterial RNA polymerase holoenzyme open promoter complex

  2015influential reference
• Structures of the RNA polymerase-σ54 reveal new and conserved regulatory strategies

  2015cited by this paper
• Structure of a bacterial RNA polymerase holoenzyme open promoter complex

  2015cited by this paper
• Structural Basis of Transcription Initiation by Bacterial RNA Polymerase Holoenzyme*

  2014cited by this paper
• Bacterial sigma factors: a historical, structural, and genomic perspective.

  2014cited by this paper
• Mycobacterial RNA polymerase forms unstable open promoter complexes that are stabilized by CarD

  2014cited by this paper
• RNA polymerase II transcription: structure and mechanism.

  2013cited by this paper
• Opening and Closing of the Bacterial RNA Polymerase Clamp

  2012cited by this paper
• Towards automated crystallographic structure refinement with phenix.refine

  2012cited by this paper
• Mechanism of transcription initiation at an activator-dependent promoter defined by single-molecule observation.

  2012cited by this paper
• Structural Basis of Transcription Initiation

  2012cited by this paper
• RELION: Implementation of a Bayesian approach to cryo-EM structure determination

  2012cited by this paper
• Structural basis of initial RNA polymerase II transcription

  2011cited by this paper
• REFMAC5 for the refinement of macromolecular crystal structures

  2011cited by this paper
• Structural basis for promoter-10 element recognition by the bacterial RNA polymerase σ subunit.

  2011cited by this paper
• A prehydrolysis state of an AAA+ ATPase supports transcription activation of an enhancer-dependent RNA polymerase

  2010cited by this paper
• One-step DNA melting in the RNA polymerase cleft opens the initiation bubble to form an unstable open complex

  2010cited by this paper
• Features and development of Coot

  2010cited by this paper
• Molecular dynamics flexible fitting: a practical guide to combine cryo-electron microscopy and X-ray crystallography.

  2009cited by this paper
• Structural basis of transcription: RNA polymerase II fidelity mechanisms and RNA 3’ fraying.

  2009influential reference
• Structural evolution of multisubunit RNA polymerases.

  2008cited by this paper
• Structural basis for transcription elongation by bacterial RNA polymerase

  2007cited by this paper
• Visualizing density maps with UCSF Chimera.

  2007cited by this paper
• Initial Transcription by RNA Polymerase Proceeds Through a DNA-Scrunching Mechanism

  2006cited by this paper
• Structural basis of transcription: role of the trigger loop in substrate specificity and catalysis.

  2006cited by this paper
• Abortive Initiation and Productive Initiation by RNA Polymerase Involve DNA Scrunching

  2006cited by this paper
• Interplay between the beta' clamp and the beta' jaw domains during DNA opening by the bacterial RNA polymerase at sigma54-dependent promoters.

  2006cited by this paper
• Interplay between the β′ Clamp and the β′ Jaw Domains during DNA Opening by the Bacterial RNA Polymerase at σ54-dependent Promoters

  2006cited by this paper
• Real-time characterization of intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase at the T7A1 promoter.

  2005cited by this paper
• Multiple sigma subunits and the partitioning of bacterial transcription space.

  2003cited by this paper
• research papers Acta Crystallographica Section D Biological

  2003cited by this paper
• Kinetic Studies and Structural Models of the Association of E. coli σ70 RNA Polymerase with the λPR Promoter: Large Scale Conformational Changes in Forming the Kinetically Significant Intermediates

  2002influential reference
• Multisubunit RNA polymerases.

  2002cited by this paper
• Kinetic studies and structural models of the association of E. coli sigma(70) RNA polymerase with the lambdaP(R) promoter: large scale conformational changes in forming the kinetically significant intermediates.

  2002cited by this paper
• The Downstream DNA Jaw of Bacterial RNA Polymerase Facilitates Both Transcriptional Initiation and Pausing*

  2002cited by this paper
• The PyMOL Molecular Graphics System

  2002cited by this paper
• The Bacterial Enhancer-Dependent ς54(ςN) Transcription Factor

  2000cited by this paper
• The sigma 54 DNA‐binding domain includes a determinant of enhancer responsiveness

  1999cited by this paper
• 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.

  1998influential reference
• Refinement of macromolecular structures by the maximum-likelihood method.

  1997cited by this paper
• In a class of its own — the RNA polymerase sigma factor σ;54 (σN)

  1993cited by this paper
• In a class of its own--the RNA polymerase sigma factor sigma 54 (sigma N).

  1993cited by this paper
• Topography of intermediates in transcription initiation of E.coli.

  1990cited by this paper

• Real-time capture of σN transcription initiation intermediates reveals mechanism of ATPase-driven activation by limited unfolding

  2025cites this paper
• Deciphering the Impact of Temperature on Pleiotropic Consequences of RNA Polymerase Mutations

  2025cites this paper
• RAD51 D-loop structures reveal the mechanism of eukaryotic RAD51-mediated strand exchange

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• Real-time capture of σN transcription initiation intermediates reveals mechanism of ATPase-driven activation by limited unfolding

  2025cites this paper
• Evidence for Stepwise Disruption of E. coli RNA Polymerase: λPR Promoter Contacts and Duplex Formation in Transcription Initiation.

  2025cites this paper
• iPro-MP: a BERT-based model to predict multiple prokaryotic promoters

  2025cites this paper
• Expanding the σ54-dependent transcription process with orthogonal designs

  2025cites this paper
• Structural basis of promoter recognition by Staphylococcus aureus RNA polymerase

  2024cites this paper
• Architecture of the spinach plastid-encoded RNA polymerase

  2024cites this paper
• Structure of the Bacteriophage PhiKZ non-virion RNA Polymerase transcribing from its promoter p119L

  2024cites this paper
• The macromolecular machine that opens up double-stranded DNA for gene transcription: from structures to functions

  2023cites this paper
• Dynamics and logic of promoter melting.

  2023cites this paper
• A general mechanism for transcription bubble nucleation in bacteria

  2023cites this paper
• Structural basis of σ54 displacement and promoter escape in bacterial transcription

  2023influential citation
• Three-Dimensional Envelope and Subunit Interactions of the Plastid-Encoded RNA Polymerase from Sinapis alba

  2022cites this paper
• Mechanisms of DNA opening revealed in AAA+ transcription complex structures

  2022cites this paper
• An Optimized Workflow for the Discovery of New Antimicrobial Compounds Targeting Bacterial RNA Polymerase Complex Formation

  2022cites this paper
• Roles of Clamp Closing and Allosteric Effects of Discriminator and Upstream Interactions on Downstream Elements in Stabilizing an E. coli RNA Polymerase-Promoter Open Complex

  2022cites this paper
• Atomic force microscopy measurements and model of DNA bending caused by binding of AraC protein

  2022cites this paper
• The archetypal gene transfer agent RcGTA is regulated via direct interaction with the enigmatic RNA polymerase omega subunit

  2022cites this paper
• Structure of the bacteriophage PhiKZ non-virion RNA polymerase

  2021cites this paper
• Universal functions of the σ finger in alternative σ factors during transcription initiation by bacterial RNA polymerase

  2021cites this paper
• Editorial: Role of Sigma Factors of RNA Polymerase in Bacterial Physiology

  2021cites this paper
• Understanding transcription across scales: From base pairs to chromosomes.

  2021cites this paper
• Transcription initiation at a consensus bacterial promoter proceeds via a “bind-unwind-load-and-lock” mechanism

  2021cites this paper
• Structure of RNA polymerase II pre-initiation complex at 2.9 Å defines initial DNA opening.

  2021cites this paper
• Temperature effects on RNA polymerase initiation kinetics reveal which open complex initiates and that bubble collapse is stepwise

  2021cites this paper
• Transcription initiation at a consensus bacterial promoter proceeds via a ‘bind-unwind-load-and-lock’ mechanism

  2021cites this paper
• Using cryo-EM to uncover mechanisms of bacterial transcriptional regulation

  2021cites this paper
• References

  2020cites this paper
• Insights into Transcriptional Repression of the Homologous Toxin-Antitoxin Cassettes yefM-yoeB and axe-txe

  2020cites this paper
• RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding

  2020cites this paper
• Kinetic-Mechanistic Evidence for Which E. coli RNA Polymerase-λPR Open Promoter Complex Initiates and for Stepwise Disruption of Contacts in Bubble Collapse

  2020cites this paper
• Structural basis of ribosomal RNA transcription regulation

  2020cites this paper
• Acclimation of bacterial cell state for high-throughput enzyme engineering using a DmpR-dependent transcriptional activation system

  2020cites this paper
• Fluorescence-Detected Conformational Changes in Duplex DNA in Open Complex Formation by E. coli RNA Polymerase: Upstream Wrapping and Downstream Bending Precede Clamp Opening and Insertion of the Downstream Duplex

  2020cites this paper
• Bacterial Enhancer Binding Proteins—AAA+ Proteins in Transcription Activation

  2020influential citation
• Regulation of transcription through the secondary channel of RNA polymerase

  2019cites this paper
• Contribution of σ70 and σN Factors to Expression of Class II pilE in Neisseria meningitidis

  2019cites this paper
• Structural basis of transcription inhibition by the DNA mimic protein Ocr of bacteriophage T7

  2019cites this paper
• Recent Advances in Understanding σ70-Dependent Transcription Initiation Mechanisms

  2019cites this paper
• Mechanisms of σ54-Dependent Transcription Initiation and Regulation

  2019influential citation
• Transcription activation in bacteria: ancient and modern.

  2019cites this paper
• Visualization of two architectures in class-II CAP-dependent transcription activation

  2019cites this paper
• Promoter Distortion and Opening in the RNA Polymerase II Cleft.

  2019cites this paper
• University of Birmingham Transcription activation in bacteria

  2019cites this paper
• The Core and Holoenzyme Forms of RNA Polymerase from Mycobacterium smegmatis

  2018cites this paper
• Region 3.2 of the σ factor controls the stability of rRNA promoter complexes and potentiates their repression by DksA

  2018cites this paper
• Structural basis of ECF-σ-factor-dependent transcription initiation

  2018influential citation
• Structure of a yeast closed complex (core CC1)

  2018cites this paper
• New insights into the adaptive transcriptional response to nitrogen starvation in Escherichia coli

  2018cites this paper