title: Visualizing translocation dynamics and nascent transcript errors in paused RNA polymerases in vivo.
abstract: BACKGROUND
  Transcription 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.
  
  
  RESULTS
  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). 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.
  
  
  CONCLUSIONS
  In 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.
authors.affiliation: Center for Cancer Research, National Cancer Institute, Frederick, MD, 21702, USA. imashimizum@mail.nih.gov.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Imashimizu
authors.affiliation: Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8673, Japan. hiroki.takahashi@chiba-u.jp.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Takahashi
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Ikoma, Nara, 630-0192, Japan. taku@bs.naist.jp.
authors.forename: Taku
authors.initials: T
authors.lastname: Oshima
authors.affiliation: Center for Cancer Research, National Cancer Institute, Frederick, MD, 21702, USA. mcintoshc@mail.nih.gov.
authors.forename: Carl
authors.initials: C
authors.lastname: McIntosh
authors.affiliation: Center for Cancer Research, National Cancer Institute, Frederick, MD, 21702, USA. bubunenm@mail.nih.gov.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Bubunenko
authors.affiliation: Center for Cancer Research, National Cancer Institute, Frederick, MD, 21702, USA. courtd@mail.nih.gov.
authors.forename: Donald L
authors.initials: DL
authors.lastname: Court
authors.affiliation: Center for Cancer Research, National Cancer Institute, Frederick, MD, 21702, USA. kashlevm@mail.nih.gov.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Kashlev
mesh_terms: D018899:CpG Islands; D004261:DNA Replication; D016373:DNA, Antisense / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D056726:Genetic Association Studies; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D035602:Transcriptional Elongation Factors / Q000235:genetics / Q000378:metabolism; D014178:Translocation, Genetic
chemical_list: D016373:DNA, Antisense; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C079668:GreA protein, E coli; C079667:GreB protein, E coli; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C108570:beta' subunit of RNA polymerase; D012321:DNA-Directed RNA Polymerases