Defining the Status of RNA Polymerase at Promoters
Leighton J. Core,
Joshua J. Waterfall,
Daniel A. Gilchrist,
David C. Fargo,
Hojoong Kwak,
Karen Adelman,
John T. Lis
Affiliations
Leighton J. Core
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
Joshua J. Waterfall
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
Daniel A. Gilchrist
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA
David C. Fargo
Laboratory of Integrated Bioinformatics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA
Hojoong Kwak
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
Karen Adelman
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA
John T. Lis
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
Recent genome-wide studies in metazoans have shown that RNA polymerase II (Pol II) accumulates to high densities on many promoters at a rate-limited step in transcription. However, the status of this Pol II remains an area of debate. Here, we compare quantitative outputs of a global run-on sequencing assay and chromatin immunoprecipitation sequencing assays and demonstrate that the majority of the Pol II on Drosophila promoters is transcriptionally engaged; very little exists in a preinitiation or arrested complex. These promoter-proximal polymerases are inhibited from further elongation by detergent-sensitive factors, and knockdown of negative elongation factor, NELF, reduces their levels. These results not only solidify the notion that pausing occurs at most promoters, but demonstrate that it is the major rate-limiting step in early transcription at these promoters. Finally, the divergent elongation complexes seen at mammalian promoters are far less prevalent in Drosophila, and this specificity in orientation correlates with directional core promoter elements, which are abundant in Drosophila.
