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Mol Cell. 2017 Aug 3;67(3):411-422.e4. doi: 10.1016/j.molcel.2017.06.027. Epub 2017 Jul 20.

Genome-wide Single-Molecule Footprinting Reveals High RNA Polymerase II Turnover at Paused Promoters.

Author information

1
Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland. Electronic address: arnaud.krebs@fmi.ch.
2
Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland.
3
Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland; Swiss Institute of Bioinformatics, Maulbeerstrasse 66, 4058 Basel, Switzerland.
4
Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland; University of Basel, Faculty of Sciences, Petersplatz 1, 4001 Basel, Switzerland. Electronic address: dirk@fmi.ch.

Abstract

Transcription initiation entails chromatin opening followed by pre-initiation complex formation and RNA polymerase II recruitment. Subsequent polymerase elongation requires additional signals, resulting in increased residence time downstream of the start site, a phenomenon referred to as pausing. Here, we harnessed single-molecule footprinting to quantify distinct steps of initiation in vivo throughout the Drosophila genome. This identifies the impact of promoter structure on initiation dynamics in relation to nucleosomal occupancy. Additionally, perturbation of transcriptional initiation reveals an unexpectedly high turnover of polymerases at paused promoters-an observation confirmed at the level of nascent RNAs. These observations argue that absence of elongation is largely caused by premature termination rather than by stable polymerase stalling. In support of this non-processive model, we observe that induction of the paused heat shock promoter depends on continuous initiation. Our study provides a framework to quantify protein binding at single-molecule resolution and refines concepts of transcriptional pausing.

KEYWORDS:

DNA footprinting; GTF; TBP; genomics; single molecule; transcription; transcriptional pausing

PMID:
28735898
PMCID:
PMC5548954
DOI:
10.1016/j.molcel.2017.06.027
[Indexed for MEDLINE]
Free PMC Article

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