These paused polymerases allow a rapid transcription response to environmental stimuli and are used during development in higher eukaryotes. For example, eukaryotic RNAPII tends to pause in a region located ≤100 bp downstream of a transcription start site, and is controlled by accessory protein factors such as NELF/DSIF. Many regulatory events derived from pausing appear to be localized in promoter-proximal regions in eukaryotes or the 5′ untranslated region (UTR) of mRNA genes in prokaryotes. In prokaryotes, the RNAP pausing/anti-pausing system that utilizes RfaH protein controls expression of genes involved in DNA transfer and virulence. Regulation of elongation via pausing has a variety of physiological consequences. Interaction of RNAP with some of these sequences results in transcriptional pausing, which occurs on average every 100 bp of transcribed DNA in vitro. RNA polymerase (RNAP) transcribes DNA of different structural and chemical sequences. CpG DNA sequences induce transcriptional pausing and G-to-A errors. coli, robust transcriptional pausing involves RNAP interaction with G-dC at the upstream end of the RNA-DNA hybrid, which interferes with translocation. 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. Gre factors efficiently proofread the errors and rescue the backtracked complexes. 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. Thus, the G-dC base pair can induce pausing in post-translocated, pre-translocated, and backtracked states of RNAP. The distance between the 5′ G-dC base pair and the 3′ end of RNA fluctuates over a three-nucleotide width. We reveal that the G-dC base pair at the 5′ end of the RNA-DNA hybrid interferes with RNAP translocation. ![]() ![]() We 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). Mechanisms of pausing associated with translocation blocks and misincorporation have been characterized in vitro, but not in vivo. Transcription errors also induce prolonged pausing, which can lead to a destabilized genome by interfering with DNA replication. Transcription elongation is frequently interrupted by pausing signals in DNA, with downstream effects on gene expression.
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