Kinetics of promoter Pol II on Hsp70 reveal stable pausing and key insights into its regulation

Martin S. Buckley; Hojoong Kwak; Warren R. Zipfel; John T. Lis

doi:10.1101/gad.231886.113

Kinetics of promoter Pol II on Hsp70 reveal stable pausing and key insights into its regulation

¹Department of Molecular Biology and Genetics,
²Department of Biomedical Engineering, Cornell University, Ithaca, New York 14853, USA

Abstract

The kinetics with which promoter-proximal paused RNA polymerase II (Pol II) undergoes premature termination versus productive elongation is central to understanding underlying mechanisms of metazoan transcription regulation. To assess the fate of Pol II quantitatively, we tracked photoactivatable GFP-tagged Pol II at uninduced Hsp70 on polytene chromosomes and showed that Pol II is stably paused with a half-life of 5 min. Biochemical analysis of short nascent RNA from Hsp70 reveals that this half-life is determined by two comparable rates of productive elongation and premature termination of paused Pol II. Importantly, heat shock dramatically increases elongating Pol II without decreasing termination, indicating that regulation acts at the step of paused Pol II entry to productive elongation.

Keywords

Footnotes

↵3 These authors contributed equally to this work.
↵4 Corresponding author

E-mail jtl10{at}cornell.edu
Supplemental material is available for this article.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.231886.113.

Received September 30, 2013.
Accepted December 3, 2013.

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