Open complex DNA scrunching: A key to transcription start site selection and promoter escape

Winkelman, Jared T.; Gourse, Richard L.

doi:10.1002/bies.201600193

Cited by 26 publications

(23 citation statements)

References 43 publications

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“…Scrunching has been observed in initiation (Winkelman and Gourse, 2017) and elongation (Zhilina et al, 2012;Strobel and Roberts, 2014) complexes in which the r-DNA contacts stabilize a scrunched intermediate. During initial rounds of RNA synthesis, the front end of RNAP moves forward as nucleotides are added to the chain, but the rear end is held in place by r-DNA contacts and the unwound DNA is pushed into the active site channel (Winkelman and Gourse, 2017). These interactions must be broken to allow escape from a promoter or from r-dependent pause induced by a 210-like sequence.…”

Section: Rfah-bound Ecs Are Not Scrunchedmentioning

confidence: 99%

“…These interactions must be broken to allow escape from a promoter or from r-dependent pause induced by a 210-like sequence. Stress accumulated during scrunching can also be relieved by release of a short abortive RNA (Winkelman and Gourse, 2017) or by backtracking, followed by cleavage of the nascent RNA and restart (Zhilina et al, 2012;Strobel and Roberts, 2014). Like r, RfaH binds to the b 0 CH and the NT strand (Sevostyanova et al, 2008) and induces a pause downstream of the ops element (Belogurov et al, 2010), suggesting that it could stabilize a scrunched intermediate.…”

Section: Rfah-bound Ecs Are Not Scrunchedmentioning

confidence: 99%

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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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Section: Rfah-bound Ecs Are Not Scrunchedmentioning

confidence: 99%

Section: Rfah-bound Ecs Are Not Scrunchedmentioning

confidence: 99%

Locking the nontemplate DNA to control transcription

Nedialkov

Svetlov

Belogurov

et al. 2018

Molecular Microbiology

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“…A growing body of evidence supports a key role of the NT DNA in the regulation of transcription. NT DNA contacts to the β and σ subunits (29,30) determine the structure and stability of promoter complexes, control start site selection, and mediate the efficiency of promoter escape, in part by modulating DNA scrunching (19,(33)(34)(35). Upon promoter escape and σ release, the NT DNA loses contacts with RNAP (17), except for transient interactions with β that control elongation and pausing (19,23,36).…”

Section: Specific Recognition Of Ops By Rfah Despite Low Sequence Idmentioning

confidence: 99%

The universally-conserved transcription factor RfaH is recruited to a hairpin structure of the non-template DNA strand

Zuber

Schweimer

NandyMazumdar

et al. 2018

Preprint

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21RfaH, a transcription regulator of the universally conserved NusG/Spt5 family, utilizes a unique 22 mode of recruitment to elongating RNA polymerase to activate virulence genes. RfaH function 23 depends critically on an ops sequence, an exemplar of a consensus pause, in the non-template 24 DNA strand of the transcription bubble. We used structural and functional analyses to elucidate 25 the role of ops in RfaH recruitment. Our results demonstrate that ops induces pausing to facilitate 26 RfaH binding and establishes direct contacts with RfaH. Strikingly, the non-template DNA forms 27 a hairpin in the RfaH:ops complex structure, flipping out a conserved T residue that is 28 specifically recognized by RfaH. Molecular modeling and genetic evidence support the notion 29 that ops hairpin is required for RfaH recruitment. We argue that both the sequence and the 30 structure of the non-template strand are read out by transcription factors, expanding the 31 repertoire of transcriptional regulators in all domains of life. 32 33 72 (Kohler et al., 2017; Saxena et al., 2018). Following TEC dissociation, RfaH has been proposed 73 to regain the autoinhibited state (Tomar et al., 2013), thus completing the cycle. 74 A model of E. coli RfaH bound to Thermus thermophilus TEC was constructed by arbitrarily 75 threading the NT DNA (absent in the X-ray structure) through the TEC (Belogurov et al., 2007). 76 While subsequent functional analysis of RfaH supports this model (Belogurov et al., 2010), the 77 path of the NT DNA and the details of ops:RfaH interactions remain unclear. The NT DNA is 78 flexible in the TEC (Kang et al., 2017) and could be trapped in a state incompatible with 79 5 productive elongation; RfaH/NusG and yeast Spt5 have been proposed to constrain the NT 80 strand to increase processivity (Crickard et al., 2016; NandyMazumdar et al., 2016). Direct 81 contacts to the NT DNA have been demonstrated recently for Bacillus subtilis NusG (Yakhnin et 82 al., 2016) and Saccharomyces cerevisiae Spt5 (Crickard et al., 2016). 83 Here we combined structural and functional analyses to dissect RfaH:ops interactions. Our data 84 argue that ops plays two roles in RfaH recruitment: it halts RNAP to aid loading of RfaH and 85 makes specific contacts with RfaH-NTD. Strikingly, we found that a small hairpin extruded from 86 the NT DNA is required for RfaH recruitment, demonstrating how NT DNA flexibility could be 87 harnessed for transcriptional regulation in this and potentially many other systems. 88 127 pausing (Figure 2D and Figure 2 -figure supplement 1), consistent with their variability in the 128 consensus pause sequence (Figure 2A). Conversely, the G12C substitution eliminated the pause 129 at U11, making measurements of RfaH antipausing activity unreliable, but did not abrogate RfaH 130 recruitment (Figures 2C,D), suggesting that pausing at U11 is dispensable for RfaH binding 131 when RNAP is transcribing slowly. 132 Observations that RfaH is recruited to RNAP transcribing the G12C template raised a possibi...

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“…Lastly, we extracted the backgrounds from non-promoter, intergenic regions of the E. coli genome that vary in GC content. Although sequences within the promoter background have been previously shown to modulate expression [44][45][46][47][48] , we varied this sequence primarily as a control to see how consistent observations were between sequence contexts. In addition to our library of σ70 promoter variants, we included 470 negative controls, which are intergenic regions that appear to be transcriptionally quiescent in RNA-Seq studies [49][50][51] .This library was synthesized, cloned, and integrated the library using this RMCE method into the nth-ydgR locus in the same direction as the DNA replication ( Figure 2B).…”

Section: Reporter and Library Design Construction And Testingmentioning

confidence: 99%

“…The background sequences as we define them include many other regions known to affect transcription, including the discriminator and initial transcribed region 26,[44][45][46][47][48] . We found modest differences in the distribution of expression for each background, and this appeared to be unrelated to background GC content ( Figure 6A).…”

Section: Identifying Complex Interactions Between Sequence Elementsmentioning

confidence: 99%

Systematic Dissection of Sequence Elements Controlling σ70 Promoters Using a Genomically-Encoded Multiplexed Reporter Assay inE. coli

Urtecho

Tripp

Insigne

et al. 2017

Preprint

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Abstract:Promoters are the key drivers of gene expression and are largely responsible for the regulation of cellular responses to time and environment. In E. coli , decades of studies have revealed most, if not all, of the sequence elements necessary to encode promoter function. Despite our knowledge of these motifs, it is still not possible to predict the strength and regulation of a promoter from primary sequence alone. Here we develop a novel multiplexed assay to study promoter function in E. coli by building a site-specific genomic recombination-mediated cassette exchange (RMCE) system that allows for the facile construction and testing of large libraries of genetic designs integrated into precise genomic locations. We build and test a library of 10,898 σ70 promoter variants consisting of all combinations of a set of eight -35 elements, eight -10 elements, three UP elements, eight spacers, and eight backgrounds. We find that the -35 and -10 sequence elements can explain approximately 74% of the variance in promoter strength within our dataset using a simple log-linear statistical model. Simple neural network models explain greater than 95% of the variance in our dataset by capturing nonlinear interactions with the spacer, background, and UP elements.

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Open complex DNA scrunching: A key to transcription start site selection and promoter escape

Cited by 26 publications

References 43 publications

Locking the nontemplate DNA to control transcription

Locking the nontemplate DNA to control transcription

The universally-conserved transcription factor RfaH is recruited to a hairpin structure of the non-template DNA strand

Systematic Dissection of Sequence Elements Controlling σ70 Promoters Using a Genomically-Encoded Multiplexed Reporter Assay inE. coli

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