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Elife. 2019 Apr 25;8. pii: e41461. doi: 10.7554/eLife.41461.

Reporter-ChIP-nexus reveals strong contribution of the Drosophila initiator sequence to RNA polymerase pausing.

Author information

1
Stowers Institute for Medical Research, Kansas City, United States.
2
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, United States.

Abstract

RNA polymerase II (Pol II) pausing is a general regulatory step in transcription, yet the stability of paused Pol II varies widely between genes. Although paused Pol II stability correlates with core promoter elements, the contribution of individual sequences remains unclear, in part because no rapid assay is available for measuring the changes in Pol II pausing as a result of altered promoter sequences. Here, we overcome this hurdle by showing that ChIP-nexus captures the endogenous Pol II pausing on transfected plasmids. Using this reporter-ChIP-nexus assay in Drosophila cells, we show that the pausing stability is influenced by downstream promoter sequences, but that the strongest contribution to Pol II pausing comes from the initiator sequence, in which a single nucleotide, a G at the +2 position, is critical for stable Pol II pausing. These results establish reporter-ChIP-nexus as a valuable tool to analyze Pol II pausing.

KEYWORDS:

D. melanogaster; Kc167 cells; RNA polymerase II pausing; TFIID; chromosomes; core promoter element; gene expression; reporter-ChIP-nexus

Conflict of interest statement

WS, SA No competing interests declared, JZ J.Z. owns a patent on ChIP-nexus. EP3234199A1

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