RNA Polymerase Pausing during Initial Transcription

Summary In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause (“initiation pause,” ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA.

• Publication year

  2016

• Venue

  Molecules and Cells

• Publication date

  2016-09-15

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1016/j.molcel.2016.08.011PMID 27618490PMCID 5031556
• 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.

• E. Coli RNA Polymerase Pauses during Initial Transcription

  2016cited by this paper
• Monitoring translocation of multisubunit RNA polymerase along the DNA with fluorescent base analogues.

  2015influential reference
• Crystal structures of the E. coli transcription initiation complexes with a complete bubble.

  2015influential reference
• Structural Basis of Transcription Initiation by Bacterial RNA Polymerase Holoenzyme*

  2014influential reference
• Interactions between RNA polymerase and the “core recognition element” counteract pausing

  2014cited by this paper
• Distinct functions of the RNA polymerase σ subunit region 3.2 in RNA priming and promoter escape

  2014influential reference
• RNA polymerase pausing and nascent RNA structure formation are linked through clamp domain movement

  2014influential reference
• A pause sequence enriched at translation start sites drives transcription dynamics in vivo

  2014cited by this paper
• The transcription bubble of the RNA polymerase-promoter open complex exhibits conformational heterogeneity and millisecond-scale dynamics: implications for transcription start-site selection.

  2013cited by this paper
• X-ray Crystal Structure of Escherichia coli RNA Polymerase σ70 Holoenzyme*

  2013influential reference
• Structure and function of the initially transcribing RNA polymerase II–TFIIB complex

  2012cited by this paper
• Structural Basis of Transcription Initiation

  2012influential reference
• Capturing reaction paths and intermediates in Cre-loxP recombination using single-molecule fluorescence

  2012cited by this paper
• Characterization of dark quencher chromophores as nonfluorescent acceptors for single-molecule FRET.

  2012cited by this paper
• Mechanism of bacterial transcription initiation: RNA polymerase - promoter binding, isomerization to initiation-competent open complexes, and initiation of RNA synthesis.

  2011cited by this paper
• Single-molecule studies of RNA polymerase: one singular sensation, every little step it takes.

  2011cited by this paper
• Sensing DNA opening in transcription using quenchable Förster resonance energy transfer.

  2010cited by this paper
• Defining the limits of single-molecule FRET resolution in TIRF microscopy.

  2010cited by this paper
• Mechanism of sequence-specific pausing of bacterial RNA polymerase

  2009cited by this paper
• Monitoring abortive initiation.

  2009influential reference
• Analysis of Promoter Targets for Escherichia coli Transcription Elongation Factor GreA In Vivo and In Vitro

  2007cited by this paper
• Structural basis for converting a general transcription factor into an operon-specific virulence regulator.

  2007cited by this paper
• Acknowledgments

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

  2006cited by this paper
• Initial Transcription by RNA Polymerase Proceeds Through a DNA-Scrunching Mechanism

  2006cited by this paper
• Sequence-resolved detection of pausing by single RNA polymerase molecules.

  2006cited by this paper
• Initial transcribed sequence mutations specifically affect promoter escape properties.

  2006cited by this paper
• Region 3.2 of the σ Subunit Contributes to the Binding of the 3′-Initiating Nucleotide in the RNA Polymerase Active Center and Facilitates Promoter Clearance during Initiation*

  2006influential reference
• Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes.

  2006cited by this paper
• Direct Modulation of RNA Polymerase Core Functions by Basal Transcription Factors

  2005cited by this paper
• Retention of transcription initiation factor sigma70 in transcription elongation: single-molecule analysis.

  2005cited by this paper
• Alternating-laser excitation of single molecules.

  2005cited by this paper
• Promoter unwinding and promoter clearance by RNA polymerase: detection by single-molecule DNA nanomanipulation.

  2004cited by this paper
• The sigma 70 subunit of RNA polymerase mediates a promoter-proximal pause at the lac promoter.

  2004influential reference
• The σ70 subunit of RNA polymerase mediates a promoter-proximal pause at the lac promoter

  2004influential reference
• Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules

  2004cited by this paper
• The sigma 70 subunit of RNA polymerase induces lacUV5 promoter-proximal pausing of transcription.

  2004cited by this paper
• Bacterial RNA polymerases: the wholo story.

  2003cited by this paper
• Cooperation Between RNA Polymerase Molecules in Transcription Elongation

  2003cited by this paper
• Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 Å resolution

  2002cited by this paper
• Promoter clearance and escape in prokaryotes.

  2002influential reference
• Structural Basis of Transcription Initiation: RNA Polymerase Holoenzyme at 4 Å Resolution

  2002influential reference
• Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex.

  2002cited by this paper
• Structural mechanism for rifampicin inhibition of bacterial rna polymerase.

  2001cited by this paper
• Translocation of sigma(70) with RNA polymerase during transcription: fluorescence resonance energy transfer assay for movement relative to DNA.

  2001cited by this paper
• A branched pathway in the early stage of transcription by Escherichia coli RNA polymerase.

  1996cited by this paper
• Escherichia coli transcript cleavage factors GreA and GreB stimulate promoter escape and gene expression in vivo and in vitro.

  1995influential reference
• GreA-induced transcript cleavage in transcription complexes containing Escherichia coli RNA polymerase is controlled by multiple factors, including nascent transcript location and structure.

  1994cited by this paper
• Kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter. Evidence for a sequential mechanism involving three steps.

  1985influential reference
• Abortive initiation and long ribonucleic acid synthesis.

  1981cited by this paper
• Productive and abortive initiation of transcription in vitro at the lac UV5 promoter.

  1980influential reference
• Cycling of ribonucleic acid polymerase to produce oligonucleotides during initiation in vitro at the lac UV5 promoter.

  1980cited by this paper
• On the mechanism of rifampicin inhibition of RNA synthesis.

  1978cited by this paper

• Structural basis of pausing during transcription initiation in mycobacterium tuberculosis

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• Single-molecule experiments reveal NusG displaces transcription initiation factor σ70 from mature elongation complexes

  2026cites this paper
• Structural insights into σ28-dependent transcription initiation and its regulation by anti-sigma factor in Pseudomonas aeruginosa

  2026cites this paper
• The displacement of the σ70 finger in initial transcription is highly heterogeneous and promoter-dependent

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• Quantitative parameters of productive transcription on T5 N25–based promoters are modulated by the initial transcribed sequence and template supercoiling

  2025cites this paper
• Unraveling single-molecule reactions via multiplexed in-situ DNA sequencing

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• Single-Molecule Imaging Reveals Transcription-Driven Supercoiling in Unconstrained DNA

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

  2025cites this paper
• W2V-repeated index: Prediction of enhancers and their strength based on repeated fragments.

  2024cites this paper
• Single-molecule reconstruction of eukaryotic factor-dependent transcription termination

  2024cites this paper
• Single-Molecule Approaches to Study DNA Condensation.

  2024cites this paper
• Towards a rational approach to promoter engineering: understanding the complexity of transcription initiation in prokaryotes

  2024influential citation
• Correlative Escherichia coli Transcription Rate and Bubble Conformation Remodeled by NusA and NusG.

  2023cites this paper
• The displacement of the σ70 finger in initial transcription is highly heterogeneous and promoter-dependent

  2023influential citation
• Beyond the approved: target sites and inhibitors of bacterial RNA polymerase from bacteria and fungi.

  2022cites this paper
• Tetrameric UvrD helicase is located at the E. coli replisome

  2022cites this paper
• Step-by-Step Regulation of Productive and Abortive Transcription Initiation by Pyrophosphorolysis.

  2022cites this paper
• Transient DNA binding to gapped DNA substrates links DNA sequence to the single-molecule kinetics of protein-DNA interactions

  2022cites this paper
• Tetrameric UvrD helicase is located at the E. coli replisome due to frequent replication blocks

  2021cites this paper
• Real-Time Single-Molecule Studies of RNA Polymerase–Promoter Open Complex Formation Reveal Substantial Heterogeneity Along the Promoter-Opening Pathway

  2021influential citation
• Mars, a molecule archive suite for reproducible analysis and reporting of single-molecule properties from bioimages

  2021cites this paper
• Watching the bacterial RNA polymerase transcription reaction by time-dependent soak-trigger-freeze X-ray crystallography.

  2021cites this paper
• Quantitative parameters of bacterial RNA polymerase open-complex formation, stabilization and disruption on a consensus promoter

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

  2021cites this paper
• Probing steps in DNA transcription using single-molecule methods

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

  2021cites this paper
• The formation of the bacterial RNA polymerase–promoter open complex involves a branched pathway

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  2020cites this paper
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  2020cites this paper
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  2020cites this paper
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  2020cites this paper
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  2020cites this paper
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  2020cites this paper
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  2020cites this paper
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  2020cites this paper
• Anti-pausing activity of region 4 of the RNA polymerase σ subunit and its regulation by σ-remodeling factors

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  2020cites this paper
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  2020cites this paper
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  2020cites this paper
• The Context-Dependent Influence of Promoter Sequence Motifs on Transcription Initiation Kinetics and Regulation

  2020cites this paper
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  2020cites this paper
• Thermus thermophilus initial transcription complex comprising sigma A and 5'-triphosphate RNA of 4 nt

  2020cites this paper
• 1 Control of transcription initiation by biased thermal 2 fluctuations on repetitive genomic sequences 3

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  2019cites this paper
• A Single-Molecule View on Cellular and Viral RNA Synthesis

  2019cites this paper
• Confinement-Free Wide-Field Ratiometric Tracking of Single Fluorescent Molecules

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  2019cites this paper
• Nonspecific toxicities of streptococcus pyogenes and Staphylococcus aureus dCas9 in Chlamydia trachomatis.

  2019cites this paper
• XACT-seq comprehensively defines the promoter-position and promoter-sequence determinants for initial-transcription pausing

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• RNA extension drives a stepwise displacement of an initiation-factor structural module in initial transcription

  2019cites this paper
• The Dynamic Landscape of Transcription Initiation in Yeast Mitochondria

  2019cites this paper
• Structural basis for transcription initiation by bacterial ECF σ factors

  2019cites this paper
• Delayed inhibition mechanism for secondary channel factor regulation of ribosomal RNA transcription

  2019cites this paper
• Regulation of asymmetries in the kinetics and protein numbers of bacterial gene expression.

  2019cites this paper
• Crystal structure of Mycobacterium tuberculosis transcription initiation complex(ECF sigma factor L) containing 5nt RNA with 6nt spacer

  2019cites this paper
• Closing and opening of the RNA polymerase trigger loop

  2019cites this paper
• RNA Polymerase: Step-by-Step Kinetics and Mechanism of Transcription Initiation.

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

  2019cites this paper
• Real-time analysis of single influenza virus replication complexes reveals large promoter-dependent differences in initiation dynamics

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• The Role of Pyrophosphorolysis in the Initiation-to-Elongation Transition by E. coli RNA Polymerase.

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

  2019cites this paper
• CarD and RbpA modify the kinetics of initial transcription and slow promoter escape of the Mycobacterium tuberculosis RNA polymerase

  2019cites this paper
• Structures and mechanism of transcription initiation by bacterial ECF factors

  2019cites this paper
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  2019cites this paper
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  2019cites this paper
• Mycobacterium tuberculosis RNA polymerase holoenzyme with ECF sigma factor sigma H

  2019cites this paper
• Mechanisms of Transcriptional Pausing in Bacteria.

  2019cites this paper
• The calculation of transcript flux ratios reveals single regulatory mechanisms capable of activation and repression

  2018cites this paper
• RbpA relaxes promoter selectivity of M. tuberculosis RNA polymerase

  2018cites this paper
• The RNA polymerase clamp interconverts dynamically among three states and is stabilized in a partly closed state by ppGpp

  2018influential citation
• Structural basis of ECF-σ-factor-dependent transcription initiation

  2018cites this paper
• DNA template sequence control of bacterial RNA polymerase escape from the promoter

  2018cites this paper
• Effects of mRNA Degradation and Site-Specific Transcriptional Pausing on Protein Expression Noise.

  2018cites this paper
• Reading of the non‐template DNA by transcription elongation factors

  2018cites this paper
• Life under the Microscope: Single-Molecule Fluorescence Highlights the RNA World.

  2018cites this paper
• Pausing controls branching between productive and non-productive pathways during initial transcription in bacteria

  2018cites this paper
• Analysis of Bacterial Transcription by "Massively Systematic Transcript End Readout," MASTER.

  2018cites this paper
• Active site closure stabilizes the backtracked state of RNA polymerase

  2018cites this paper
• Studying transcription initiation by RNA polymerase with diffusion‐based single‐molecule fluorescence

  2017cites this paper
• The effect of macromolecular crowding on single-round transcription by Escherichia coli RNA polymerase

  2017cites this paper
• Pausing controls branching between productive and non-productive pathways during initial transcription in bacteria

  2017cites this paper
• Single-molecule and super-resolution imaging of transcription in living bacteria

  2017cites this paper
• Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes

  2017cites this paper
• Possible roles of σ-dependent RNA polymerase pausing in transcription regulation

  2017cites this paper
• Optical tweezers studies of transcription by eukaryotic RNA polymerases

  2017cites this paper
• Deep sequencing approaches for the analysis of prokaryotic transcriptional boundaries and dynamics.

  2017cites this paper
• Interplay between σ region 3.2 and secondary channel factors during promoter escape by bacterial RNA polymerase.

  2017cites this paper
• How to switch the motor on: RNA polymerase initiation steps at the single‐molecule level

  2017cites this paper
• Different types of pausing modes during transcription initiation

  2017influential citation
• Combinatorial effect of promoter activity, mRNA degradation and site-specific transcriptional pausing in modulating protein expression noise

  2017cites this paper
• Functional interactions of the Transcription Factor B during transcription initiation in Pyrococcus furiosus

  2017influential citation
• A link between transcription fidelity and pausing in vivo

  2017cites this paper
• Mechanism of transcription initiation and promoter escape by E. coli RNA polymerase

  2017cites this paper
• Structural basis of transcription inhibition by fidaxomicin (lipiarmycin A3)

  2017influential citation
• Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase

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

  2016cites this paper
• Single-molecule FRET studies in live bacteria

  2015cites this paper