Structural and mechanistic basis of σ-dependent transcriptional pausing

In σ-dependent transcriptional pausing, the transcription initiation factor σ, translocating with RNA polymerase (RNAP), makes sequence-specific protein-DNA interactions with a promoter-like sequence element in the transcribed region, inducing pausing. It has been proposed that, in σ-dependent pausing, the RNAP active center can access off-pathway “backtracked” states that are substrates for the transcript-cleavage factors of the Gre family, and on-pathway “scrunched” states that mediate pause escape. Here, using site-specific protein-DNA photocrosslinking to define positions of the RNAP trailing and leading edges and of σ relative to DNA at the λPR’ promoter, we show directly that σ-dependent pausing in the absence of GreB in vitro predominantly involves a state backtracked by 2-4 bp, and that σ-dependent pausing in the presence of GreB in vitro and in vivo predominantly involves a state scrunched by 2-3 bp. Analogous experiments with a library of 47 (∼16,000) transcribed-region sequences show that the state scrunched by 2-3 bp--and only that state--is associated with the consensus sequence, T-3N-2Y-1G+1, (where -1 corresponds to the position of the RNA 3’ end), which is identical to the consensus for pausing in initial transcription, and which is related to the consensus for pausing in transcription elongation. Experiments with heteroduplex templates show that sequence information at position T-3 resides in the DNA nontemplate strand. A cryo-EM structure of a complex engaged in σ-dependent pausing reveals positions of DNA scrunching on the DNA nontemplate and template strands and suggests that position T-3 of the consensus sequence exerts its effects by facilitating scrunching

Structural and mechanistic basis of σ-dependent transcriptional pausing

SignificanceThe paradigmatic example of factor-dependent pausing in transcription elongation is σ-dependent pausing, in which sequence-specific σ-DNA interaction with a - 10 element-like sequence in a transcribed region results in pausing of a σ-containing transcription elongation complex. It has been proposed that σ-dependent pausing involves DNA scrunching, and that sequences downstream of the -10 element-like sequence modulate DNA scrunching. Here, using site-specific protein-DNA photocrosslinking, high-throughput sequencing, and cryoelectron microscopy structure determination, we show directly that σ-dependent pausing involves DNA scrunching, we define a consensus sequence for formation of a stable scrunched paused complex that is identical to the consensus sequence for pausing in initial transcription, and we identify positions of DNA scrunching on DNA nontemplate and template strands. Our results illuminate the structural and mechanistic basis of σ-dependent transcriptional pausing

Detection of PDI in <i>V</i>. <i>cholera</i>.

<p>A-D. Analysis of PDI by 5′ RNA-seq. Percentage of transcripts emanating from position −1 of uTSRs (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005348#pgen.1005348.s003" target="_blank">S2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005348#pgen.1005348.s004" target="_blank">S3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005348#pgen.1005348.s005" target="_blank">S4</a> Tables) with the indicated sequence at −1 and +1 in cells carrying wild-type concentrations of 2- to ~4-nt RNAs (wild-type) or cells in which the oligoRNase NrnB was ectopically expressed (Nrn). The Nrn effect reported in panels A and C represents the difference in these values. uTSRs with above average Nrn effect are highlighted in black. The total number of uTSRs used to calculate the percentages is indicated. Values in panels A and B are calculated from biological replicates listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005348#pgen.1005348.s003" target="_blank">S2 Table</a>, values in panel C is calculated from replicates listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005348#pgen.1005348.s004" target="_blank">S3 Table</a> and values in panel D are calculated from replicates listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005348#pgen.1005348.s005" target="_blank">S4 Table</a>. exp, exponential phase; sta, stationary phase; all, analysis of all 5′ ends; ppp, analysis of 5′ triphosphate ends; ppp+OH, analysis of 5′ triphosphate ends and 5′ hydroxyl ends; ppp+p, analysis of 5′ triphosphate ends and 5′ monophosphate ends. E. and F. Analysis of PDI at the promoters associated with <i>VC1904</i> (pVC1904) and <i>VCA0783</i> (p<i>VCA0783</i>). Top of each panel shows promoter sequence. Indicated are positions +1, −1 and the promoter −10 and −35 elements. Middle of each panel shows results of 5′ RNA-seq. Graphs on left show average distribution of all 5′ ends between positions −3 and +4 in cells carrying wild-type concentrations of 2- to ~4-nt RNAs (wt) or cells in which the oligoRNase NrnB was ectopically expressed (Nrn) as detected by 5′ RNA-seq during stationary phase. Graphs on the right show average distribution of all 5′ ends between positions −3 and +4 for p<i>VCA0783</i> in wild-type cells during stationary phase (sta) or exponential phase (exp). Bottom of each panel shows primer extension analysis of plasmid-borne promoter variants carrying the indicated sequences at positions −1/+1 (left) or primer extension analysis of chromosomally encoded promoters during exponential phase (exp) or stationary phase (sta).</p

Detection of PDI in <i>E</i>. <i>coli</i>.

<p>A. Analysis of PDI by 5′ RNA-seq. Percentage of transcripts emanating from position −1 of uTSRs with the indicated sequence at −1 and +1 in cells carrying wild-type concentrations of 2- to ~4-nt RNAs (wild-type) or cells in which the oligoRNase NrnB was ectopically expressed (Nrn). The Nrn effect represents the difference in these values. uTSRs with above average Nrn effect are highlighted in black. The total number of uTSRs used to calculate the percentages is indicated. Values are calculated from biological replicates listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005348#pgen.1005348.s009" target="_blank">S8 Table</a>. Data is derived from the analysis of all 5′ ends during stationary phase. B. Analysis of PDI at the promoter associated with <i>tomB</i>, p<i>tomB</i>, by 5′ RNA-seq. Sequence of p<i>tomB</i> is shown. Indicated are positions +1, −1 and the promoter −10 and −35 elements. Graph on the left shows average distribution of 5′ ends between positions −3 and +4 for p<i>tomB</i> in cells carrying wild-type concentrations of 2- to ~4-nt RNAs or cells in which the oligoRNase NrnB was ectopically expressed (Nrn) as detected by 5′ RNA-seq analysis of all 5′ ends during stationary phase. Graph on the right shows the average distribution of 5′ ends between positions −3 and +4 for p<i>tomB</i> in cells carrying wild-type concentrations of 2- to ~4-nt RNAs as detected by 5′ RNA-seq analysis of hydroxyl 5′ ends (OH), monophosphate 5′ ends (P), or triphosphate 5′ ends (PPP) during stationary phase. C. Primer extension analysis of plasmid-borne p<i>tomB</i> variants carrying the indicated sequence at −1 and +1 in cells carrying wild-type concentrations of 2- to ~4-nt RNAs (wt) or cells in which the oligoRNase NrnB was ectopically expressed (Nrn). D. Primer extension analysis of the plasmid-borne p<i>tomB</i> variant carrying the sequence G₋₁G₊₁ during exponential phase (exp) or stationary phase (sta).</p