Visualizing translocation dynamics and nascent transcript errors in paused RNA polymerases in vivo

Abstract

BackgroundTranscription elongation is frequently interrupted by pausing signals in DNA, with downstream effects on gene expression. Transcription errors also induce prolonged pausing, which can lead to a destabilized genome by interfering with DNA replication. Mechanisms of pausing associated with translocation blocks and misincorporation have been characterized in vitro, but not in vivo.ResultsWe investigate the pausing pattern of RNA polymerase (RNAP) in Escherichia coli by a novel approach, combining native elongating transcript sequencing (NET-seq) with RNase footprinting of the transcripts (RNET-seq). We reveal that the G-dC base pair at the 5′ end of the RNA-DNA hybrid interferes with RNAP translocation. The distance between the 5′ G-dC base pair and the 3′ end of RNA fluctuates over a three-nucleotide width. Thus, the G-dC base pair can induce pausing in post-translocated, pre-translocated, and backtracked states of RNAP. Additionally, a CpG sequence of the template DNA strand spanning the active site of RNAP inhibits elongation and induces G-to-A errors, which leads to backtracking of RNAP. Gre factors efficiently proofread the errors and rescue the backtracked complexes. We also find that pausing events are enriched in the 5′ untranslated region and antisense transcription of mRNA genes and are reduced in rRNA genes.ConclusionsIn E. coli, robust transcriptional pausing involves RNAP interaction with G-dC at the upstream end of the RNA-DNA hybrid, which interferes with translocation. CpG DNA sequences induce transcriptional pausing and G-to-A errors.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0666-5) contains supplementary material, which is available to authorized users.

Control of transcriptional pausing by biased thermal fluctuations on repetitive genomic sequences

“…RNAP pause sites in the entire E. coli genome were identified by the RNETseq method, sequencing nascent RNA molecules bound by paused RNAPs from the 3′ ends (10). In this method, one can define pausing with a variable level of confidence by changing the parameter representing the minimal fraction of sequencing reads for a segment having 3′ RNA ends at any genomic positions (10).…”

“…During elongation, cognate NTP binding to the RNAP active center prevents backward motion of RNAP along DNA, which is driven by thermal fluctuations, ensuring its net forward-biased RNAP motion coupled with the elongation reaction (6). Upon pausing, such an NTP binding is blocked by a high energy barrier to forward translocation generated by the consensus PIE (10). In the absence of cognate NTP binding, RNAP diffuses upstream of PIE along DNA.…”

“…Translocation is a smooth process (6,7), except in cases where certain DNA sequences impose intrinsic translocation barriers (1,2,8). This block of translocation and any inhibition of the next bond formation are causes for RNAP pausing (1,(9)(10)(11).…”

“…To investigate the sequence motif that causes transcriptional pausing and the distribution in vivo, we have previously performed native elongating transcript sequencing (NET-seq) (22) combined with RNase footprinting of the transcripts (RNET-seq) (10). This approach identified GNNNNNNTGCG as a representative RNAP pause-inducing element (PIE) in Escherichia coli cells.…”

Transcription elongation

“…The EPS pause likely is captured in the pretranslocated state, 20 and is characterized primarily by sequence elements at the upstream (G in the RNA) and downstream (Py at the RNA pause end and G following) boundaries of the templating RNA/DNA hybrid in the enzyme; the function of these sequences is understandable primarily as energetic barriers imposed by strong dG/dC or rG/dC base pairs that must be unwound for translocation to occur, although nucleotide-specific interactions also might modulate the pause. 32 The clearest function of the EPS is as a component of compound pausing regulatory signals. In the bacteriophage lambda late gene operon, a pause is induced by binding of s70 to a variant of the promoter ¡10 sequence site located 13 nucleotides downstream of the actual promoter sequence.…”