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?38-dependent promoter-proximal pausing by bacterial RNA polymerase.

ABSTRACT: Transcription initiation by bacterial RNA polymerase (RNAP) requires a variable ? subunit that directs it to promoters for site-specific priming of RNA synthesis. The principal ? subunit responsible for expression of house-keeping genes can bind the transcription elongation complex after initiation and induce RNAP pausing through specific interactions with promoter-like motifs in transcribed DNA. We show that the stationary phase and stress response ?38 subunit can also induce pausing by Escherichia coli RNAP on DNA templates containing promoter-like motifs in the transcribed regions. The pausing depends on ?38 contacts with the DNA template and RNAP core enzyme and results in formation of backtracked transcription elongation complexes, which can be reactivated by Gre factors that induce RNA cleavage by RNAP. Our data suggest that ?38 can bind the transcription elongation complex in trans but likely acts in cis during transcription initiation, by staying bound to RNAP and recognizing promoter-proximal pause signals. Analysis of ?38-dependent promoters reveals that a substantial fraction of them contain potential pause-inducing motifs, suggesting that ?38-depended pausing may be a common phenomenon in bacterial transcription.

SUBMITTER: Petushkov I

PROVIDER: S-EPMC5389655 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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σ38-dependent promoter-proximal pausing by bacterial RNA polymerase.

Petushkov Ivan I Esyunina Daria D Kulbachinskiy Andrey A

Nucleic acids research 20170401 6

Transcription initiation by bacterial RNA polymerase (RNAP) requires a variable σ subunit that directs it to promoters for site-specific priming of RNA synthesis. The principal σ subunit responsible for expression of house-keeping genes can bind the transcription elongation complex after initiation and induce RNAP pausing through specific interactions with promoter-like motifs in transcribed DNA. We show that the stationary phase and stress response σ38 subunit can also induce pausing by Escheri ...[more]

PMID: 27928053

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Project description:Herpes simplex virus 1 (HSV-1) genes are transcribed by cellular RNA polymerase II (Pol II). Expression of viral immediate early (?) genes is followed sequentially by early (?), late (?1), and true late (?2) genes. We used precision nuclear run-on with deep sequencing to map and to quantify Pol II on the HSV-1(F) genome with single-nucleotide resolution. Approximately 30% of total Pol II relocated to viral genomes within 3?h postinfection (hpi), when it occupied genes of all temporal classes. At that time, Pol II on ? genes accumulated most heavily at promoter-proximal pause (PPP) sites located ?60?nucleotides downstream of the transcriptional start site, while ? genes bore Pol II more evenly across gene bodies. At 6 hpi, Pol II increased on ?1 and ?2 genes while Pol II pausing remained prominent on ? genes. At that time, average cytoplasmic mRNA expression from ? and ? genes decreased, relative to levels at 3 hpi, while ?1 relative expression increased slightly and ?2 expression increased more substantially. Cycloheximide treatment during the first 3?h reduced the amount of Pol II associated with the viral genome and confined most of the remaining Pol II to ? gene PPP sites. Inhibition of both cyclin-dependent kinase 9 activity and viral DNA replication reduced Pol II on the viral genome and restricted much of the remaining Pol II to PPP sites.IMPORTANCE These data suggest that viral transcription is regulated not only by Pol II recruitment to viral genes but also by control of elongation into viral gene bodies. We provide a detailed map of Pol II occupancy on the HSV-1 genome that clarifies features of the viral transcriptome, including the first identification of Pol II PPP sites. The data indicate that Pol II is recruited to late genes early in infection. Comparing ? and ? gene occupancy at PPP sites and gene bodies suggests that Pol II is released more efficiently into the bodies of ? genes than ? genes at 3 hpi and that repression of ? gene expression late in infection is mediated by prolonged promoter-proximal pausing. In addition, DNA replication is required to maintain full Pol II occupancy on viral DNA and to promote elongation on late genes later in infection.

Dataset Information

?38-dependent promoter-proximal pausing by bacterial RNA polymerase.

Publications

σ38-dependent promoter-proximal pausing by bacterial RNA polymerase.

Similar Datasets

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