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Visualizing translocation dynamics and nascent transcript errors in paused RNA polymerases in vivo.
Imashimizu, Masahiko; Takahashi, Hiroki; Oshima, Taku; McIntosh, Carl; Bubunenko, Mikhail; Court, Donald L; Kashlev, Mikhail.
Afiliación
  • Imashimizu M; Center for Cancer Research, National Cancer Institute, Frederick, MD, 21702, USA. imashimizum@mail.nih.gov.
  • Takahashi H; Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8673, Japan. hiroki.takahashi@chiba-u.jp.
  • Oshima T; Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Ikoma, Nara, 630-0192, Japan. taku@bs.naist.jp.
  • McIntosh C; Center for Cancer Research, National Cancer Institute, Frederick, MD, 21702, USA. mcintoshc@mail.nih.gov.
  • Bubunenko M; Center for Cancer Research, National Cancer Institute, Frederick, MD, 21702, USA. bubunenm@mail.nih.gov.
  • Court DL; Center for Cancer Research, National Cancer Institute, Frederick, MD, 21702, USA. courtd@mail.nih.gov.
  • Kashlev M; Center for Cancer Research, National Cancer Institute, Frederick, MD, 21702, USA. kashlevm@mail.nih.gov.
Genome Biol ; 16: 98, 2015 May 15.
Article en En | MEDLINE | ID: mdl-25976475
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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Translocación Genética / ARN Polimerasas Dirigidas por ADN / Escherichia coli Tipo de estudio: Prognostic_studies Idioma: En Revista: Genome Biol Asunto de la revista: BIOLOGIA MOLECULAR / GENETICA Año: 2015 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Reino Unido
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Translocación Genética / ARN Polimerasas Dirigidas por ADN / Escherichia coli Tipo de estudio: Prognostic_studies Idioma: En Revista: Genome Biol Asunto de la revista: BIOLOGIA MOLECULAR / GENETICA Año: 2015 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Reino Unido