RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes (original) (raw)
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Locking the nontemplate DNA to control transcription
Molecular Microbiology, 2018
Universally conserved NusG/Spt5 factors reduce RNA polymerase pausing and arrest. In a widely accepted model, these proteins bridge the RNA polymerase clamp and lobe domains across the DNA channel, inhibiting the clamp opening to promote pause-free RNA synthesis. However, recent structures of paused transcription elongation complexes show that the clamp does not open and suggest alternative mechanisms of antipausing. Among these mechanisms, direct contacts of NusG/Spt5 proteins with the nontemplate DNA in the transcription bubble have been proposed to prevent unproductive DNA conformations and thus inhibit arrest. We used Escherichia coli RfaH, whose interactions with DNA are best characterized, to test this idea. We report that RfaH stabilizes the upstream edge of the transcription bubble, favoring forward translocation, and protects the upstream duplex DNA from exonuclease cleavage. Modeling suggests that RfaH loops the nontemplate DNA around its surface and restricts the upstream DNA duplex mobility. Strikingly, we show that RfaH-induced DNA protection and antipausing activity can be mimicked by shortening the nontemplate strand in elongation complexes assembled on synthetic scaffolds. We propose that remodeling of the nontemplate DNA controls recruitment of regulatory factors and R-loop formation during transcription elongation across all life.
Initial Transcription by RNA Polymerase Proceeds Through a DNA-Scrunching Mechanism
Science, 2006
Using fluorescence resonance energy transfer to monitor distances within single molecules of abortively initiating transcription initiation complexes, we show that initial transcription proceeds through a "scrunching" mechanism, in which RNA polymerase (RNAP) remains fixed on promoter DNA and pulls downstream DNA into itself and past its active center. We show further that putative alternative mechanisms for RNAP-active-center translocation in initial transcription, involving "transient excursions" of RNAP relative to DNA or "inchworming" of RNAP relative to DNA, do not occur. The results support a model in which a stressed intermediate, with DNA-unwinding stress and DNA-compaction stress, is formed during initial transcription, and in which accumulated stress is used to drive breakage of interactions between RNAP and promoter DNA and between RNAP and initiation factors during promoter escape.
Molecular Cell, 2008
Elucidating the mechanism of transcription initiation by RNA polymerases (RNAP) is essential for understanding gene transcription and regulation. Although several models such as DNA scrunching, RNAP translation, and RNAP rotation have been proposed, the mechanism of initiation by T7 RNAP has remained unclear. Using ensemble and single molecule Förster resonance energy transfer (FRET) studies, we provide evidence for concerted DNA scrunching and rotation during initiation by T7 RNAP. A constant spatial distance between the upstream and downstream edges of initiation complexes making 4-7 nt RNA supports the DNA scrunching model, but not the RNAP translation or the pure rotation model. DNA scrunching is accompanied by moderate hinging motion (18 ± 4°) of the promoter towards the downstream DNA. The observed step-wise conformational changes provide a basis to understand abortive RNA synthesis during early stages of initiation and promoter escape during the later stages that allows transition to processive elongation.
Journal of molecular biology, 2014
Bacterial RNA polymerase (RNAP) makes extensive contacts with duplex DNA downstream of the transcription bubble in initiation and elongation complexes. We investigated the role of downstream interactions in formation of catalytically competent transcription initiation complex by measuring initiation activity of stable RNAP complexes with model promoter DNA fragments whose downstream ends extend from +3 to +21 relative to the transcription start site at +1. We found that DNA downstream of position +6 does not play a significant role in transcription initiation when RNAP-promoter interactions upstream of the transcription start site are strong and promoter melting region is AT rich. Further shortening of downstream DNA dramatically reduces efficiency of transcription initiation. The boundary of minimal downstream DNA duplex needed for efficient transcription initiation shifted further away from the catalytic center upon increasing the GC content of promoter melting region or in the pr...
Journal of Molecular Biology, 2005
Transcription initiation in bacteria requires melting of w13 bp of promoter DNA. The mechanism of the melting process is not fully understood. Escherichia coli RNA polymerase bearing a deletion of the b subunit lobe I (amino acid residues 186-433) initiates melting of the K10 promoter element but cannot propagate the melting downstream, towards the transcription initiation start site (C1). However, in the presence of nucleotides, stable downstream melting is induced. Here, we studied lacUV5 promoter complexes formed by the mutant enzyme by crosslinking RNA polymerase subunits to single-stranded DNA in the transcription bubble. In the absence of NTPs, a contact between the s 70 subunit and the non-template strand of the K10 promoter element was detected. This contact disappeared in the presence of NTPs. Instead, a new s 70 -DNA contact as well as stable b 0 and b subunit contacts with the nontemplate DNA downstream of the K10 promoter element were established. In terms of the two-step (upstream initiation/downstream propagation) model of promoter melting, our data suggest that b lobe I induces the propagation of promoter melting by directing downstream promoter DNA duplex towards the downstream DNA-binding channel (b 0 clamp). Establishment of downstream contacts leads to remodeling of upstream interactions between s 70 and the K10 promoter element that might facilitate promoter escape and s release.
The role of RNA polymerase subunit in promoter-independent initiation of transcription
Proceedings of the National Academy of Sciences, 2004
In bacteria, initiation of transcription depends on the RNA polymerase subunit, which brings catalytically proficient RNA polymerase core to promoters by binding to specific DNA elements located upstream of the transcription start point. Here, we study -dependent synthesis of a transcript that is used to prime replication of the single-stranded genome of bacteriophage M13. We show that, in this system, plays no role in DNA recognition, which is accomplished solely through RNA polymerase core interaction with DNA downstream of the transcription start point. However, is required for full-sized transcript synthesis by allowing RNA polymerase core to escape into productive elongation. RNA polymerase may play a similar role during replication primer synthesis in other bacterial mobile elements whose life cycle involves a single-stranded DNA stage.
Upstream Binding of Idling RNA Polymerase Modulates Transcription Initiation from a Nearby Promoter
Journal of Biological Chemistry, 2015
Background: The fis promoter upstream region harbors RNA polymerase binding sites of unknown function. Results: Modifications of the upstream polymerase binding affect fis gene expression in a supercoiling-dependent manner. Conclusion: Concomitant binding of RNA polymerase at the fis promoter and upstream region acts as a topological device regulating transcription. Significance: RNA polymerase can act as an architectural factor modulating the activity of transcription initiation complexes.
Cell, 2006
Regulation of transcription initiation is generally attributable to activator/repressor proteins that bind to specific DNA sequences. However, regulators can also achieve specificity by binding directly to RNA polymerase (RNAP) and exploiting the kinetic variation intrinsic to different RNAP-promoter complexes. We report here a previously unknown interaction with Escherichia coli RNAP that defines an additional recognition element in bacterial promoters. The strength of this sequence-specific interaction varies at different promoters and affects the lifetime of the complex with RNAP. Selection of rRNA promoter mutants forming long-lived complexes, kinetic analyses of duplex and bubble templates, dimethylsulfate footprinting, and zero-Angstrom crosslinking demonstrated that s subunit region 1.2 directly contacts the nontemplate strand base two positions downstream of the À10 element (within the ''discriminator'' region). By making a nonoptimal s1.2discriminator interaction, rRNA promoters create the short-lived complex required for specific responses to the RNAP binding factors ppGpp and DksA, ultimately accounting for regulation of ribosome synthesis.
Region 1.2 of the RNA polymerase σ subunit controls recognition of the −10 promoter element
The EMBO Journal, 2007
Recognition of the À10 promoter consensus element by region 2 of the bacterial RNA polymerase r subunit is a key step in transcription initiation. r also functions as an elongation factor, inducing transcription pausing by interacting with transcribed DNA non-template strand sequences that are similar to the À10 element sequence. Here, we show that the region 1.2 of Escherichia coli r 70 , whose function was heretofore unknown, is strictly required for efficient recognition of the non-template strand of À10-like pause-inducing DNA sequence by r region 2, and for r-dependent promoter-proximal pausing. Recognition of the fork-junction promoter DNA by RNA polymerase holoenzyme also requires r region 1.2 and thus resembles the pause-inducing sequence recognition. Our results, together with available structural data, support a model where r region 1.2 acts as a core RNA polymerase-dependent allosteric switch that modulates non-template DNA strand recognition by r region 2 during transcription initiation and elongation. The EMBO Journal (2007) 26, 955-964.