Structural Basis for NusA Stabilized Transcriptional Pausing

Guo, Xieyang; Myasnikov, Alexander G.; Chen, James; Crucifix, Corinne; Pápai, Gábor; Takács, Mária; Schultz, Patrick; Weixlbaumer, A.

doi:10.1016/j.molcel.2018.02.008

Cited by 164 publications

(251 citation statements)

References 64 publications

(120 reference statements)

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“…The region on the surface of RNAP around the RNA exit channel contains elements important for the stability of RNAP complexes. These elements include the x subunit (Weiss & Shaw, 2015) and the b-flap helix that interacts also with other proteins, such as NusA (Twist et al, 2011;Tagami et al, 2014;Ma et al, 2015;Guo et al, 2018). Whether and how RNase J1 interacts with these elements is currently unknown, and the details of the RNase J1-RNAP contacts will be studied in future experiments.…”

Section: Rnase J1 and Its Role In Disassembly Of Transcription Complexesmentioning

confidence: 99%

The torpedo effect inBacillus subtilis:RNase J1 resolves stalled transcription complexes

et al. 2019

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RNase J1 is the major 5 0 -to-3 0 bacterial exoribonuclease. We demonstrate that in its absence, RNA polymerases (RNAPs) are redistributed on DNA, with increased RNAP occupancy on some genes without a parallel increase in transcriptional output. This suggests that some of these RNAPs represent stalled, non-transcribing complexes. We show that RNase J1 is able to resolve these stalled RNAP complexes by a "torpedo" mechanism, whereby RNase J1 degrades the nascent RNA and causes the transcription complex to disassemble upon collision with RNAP. A heterologous enzyme, yeast Xrn1 (5 0 -to-3 0 exonuclease), is less efficient than RNase J1 in resolving stalled Bacillus subtilis RNAP, suggesting that the effect is RNase-specific. Our results thus reveal a novel general principle, whereby an RNase can participate in genome-wide surveillance of stalled RNAP complexes, preventing potentially deleterious transcription-replication collisions.

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Section: Rnase J1 and Its Role In Disassembly Of Transcription Complexesmentioning

confidence: 99%

The torpedo effect inBacillus subtilis:RNase J1 resolves stalled transcription complexes

et al. 2019

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“…First, the bridging contacts to GL do not appear to be universally important; E. coli NusG does not require GL for antipausing (NandyMazumdar et al, 2016) and antibacktracking (Turtola and Belogurov, 2016) activities. Second, in cryo-EM structures of the hairpin-stabilized paused E. coli ECs (Guo et al, 2018;Kang et al, 2018a), the clamp does not open, and instead undergoes a minor rotation termed 'swiveling'. Third, the evidence in support of the key roles of direct NTD-DNA contacts has been accumulating.…”

Section: Introductionmentioning

confidence: 99%

Locking the nontemplate DNA to control transcription

Nedialkov

Svetlov

Belogurov

et al. 2018

Molecular Microbiology

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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.

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“…Hairpin-dependent pausing involves nucleation of an RNA hairpin at the mouth of the RNAP RNA-exit channel, followed by propagation of the RNA hairpin stem and penetration of the RNA hairpin stem into the RNAP RNA-exit channel, where the RNA hairpin stem makes RNA-RNAP interactions that promote pausing ( Fig. 4B, top; [25][26][27]. RNA hairpin-dependent transcription termination--the mode of termination that is most common in bacterial and bacteriophage transcription, and the mode of termination that occurs at the terminator preceding lambdoid bacteriophage late genes--involves nucleation of an RNA hairpin at the mouth of the RNA-exit channel, followed by propagation of the RNA hairpin stem and penetration of the RNA hairpin stem into the RNAP RNA-exit channel, followed by further propagation of the RNA hairpin stem that exerts mechanical forces that extract the RNA from RNAP or cause RNAP to translocate forward off the RNA (28).…”

Section: Formation Of the Q21-loading Complex Entails Remarkable Largmentioning

confidence: 99%

“…S7). Model building indicates that interactions of Q21 H2 with the RNAP ZBD and RNAP lid inside the RNAP RNA-exit channel could sterically preclude both the ~3° swivelling associated with hairpin-dependent pausing (25)(26)(27) and the smaller, ~1.5°, swivelling associated with elemental pausing (25; referred to as "pre-swivelling" in 30; Fig. S7A-B).…”

Section: Formation Of the Q21-loading Complex Entails Remarkable Largmentioning

confidence: 99%

Structural basis of Q-dependent antitermination

Yin

Kaelber

Ebright

2019

Preprint

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One Sentence Summary:Q forms a "nozzle" that narrows the RNA polymerase RNA-exit channel and extrudes ssRNA, preventing formation of RNA hairpins. Abstract:Lambdoid bacteriophage Q protein mediates the switch from middle to late bacteriophage gene expression by enabling RNA polymerase (RNAP) to read through transcription terminators preceding bacteriophage late genes. Q loads onto RNAP engaged in promoter-proximal pausing at a Q binding element (QBE) and an adjacent sigma-dependent pause element (SDPE) to yield a "Q-loading complex,"and Q subsequently translocates with RNAP as a pausing-deficient, termination-deficient "Q-loaded complex." Here, we report high-resolution structures of four states on the pathway of antitermination by Q from bacteriophage 21 (Q21): Q21, the Q21-QBE complex, the Q21-loading complex, and the Q21-loaded complex. The results show that Q21 forms a torus--a "nozzle"--that narrows and extends the RNAP RNA-exit channel, extruding single-stranded RNA and preventing formation of pause and terminator hairpins. Main Text:Lambdoid bacteriophage Q protein regulates gene expression through transcription antitermination, a mechanism of regulation that is widely used in bacteria and bacteriophage but that has been poorly understood (1-10; reviewed in 11-13).Q mediates the temporal switch from middle to late bacteriophage gene expression by enabling RNA polymerase (RNAP) to read through a transcription terminator preceding bacteriophage late genes ( Fig. 1A; 1-13). The Q-dependent gene regulatory cassette consists of the gene for Q followed by a transcription unit comprising a promoter (PR'), a promoter-proximal σ-dependent pause element (SDPE; a sequence resembling a promoter -10 element, at which σR2, the σ-factor module that recognizes promoter -10 elements, re-establishes sequence-specific protein-DNA interactions), and a terminator, followed by bacteriophage late genes. In the absence of Q, RNAP initiating transcription at the PR' promoter pauses at the SDPE and terminates at the terminator, and late genes are not expressed; in the presence of Q, RNAP initiating at the PR' promoter, escapes the SDPE and reads through the terminator, and late genes are expressed. 2Q is targeted to the PR' transcriptional unit through a multi-step process entailing: (i) formation of a "Q-loading complex" comprising a Q protein bound to a Q binding element (QBE) and a σ-containing transcription elongation complex (TEC) paused at the SDPE, and (ii) transformation into a "Q-loaded complex" comprising a Q-containing TEC that translocates processively, ignores pause elements, and ignores terminators (Fig. 1B; 3-13).Here, we report a set of four structures that define the structural basis of antitermination by Q from lambdoid bacteriophage Q21 (Q21; 11,14-15): i.e., (i) a crystal structure of Q21, (ii) a crystal structure of the Q21-QBE complex, (iii) a cryo-EM structure of the Q-loading complex, and (4) a cryo-EM structure of the Q-loaded complex.We solved a crystal structure of Q21 at 1.9 Å resolution by use of single...

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Structural Basis for NusA Stabilized Transcriptional Pausing

Cited by 164 publications

References 64 publications

The torpedo effect inBacillus subtilis:RNase J1 resolves stalled transcription complexes

The torpedo effect inBacillus subtilis:RNase J1 resolves stalled transcription complexes

Locking the nontemplate DNA to control transcription

Structural basis of Q-dependent antitermination

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