Promoter-Proximal Pausing on the hsp70Promoter in Drosophila melanogaster Depends on the Upstream Regulator

Abstract

RNA polymerase II pauses in the promoter-proximal region of many genes during transcription. In the case of the hsp70promoter from Drosophila melanogaster, this pause is long-lived and occurs even when the gene is not induced. Paused polymerase escapes during heat shock when the transcriptional activator heat shock factor associates with the promoter. However, pausing is still evident, especially when induction is at an intermediate level. Yeast Gal4 protein (Gal4p) will induce transcription of the hsp70 promoter in Drosophila when binding sites for Gal4p are positioned upstream from the hsp70 TATA element. To further our understanding of promoter-proximal pausing, we have analyzed the effect of Gal4p on promoter-proximal pausing in salivary glands of Drosophila larvae. Using permanganate genomic footprinting, we observed that various levels of Gal4p induction resulted in an even distribution of RNA polymerase throughout the first 76 nucleotides of the transcribed region. In contrast, promoter-proximal pausing still occurs on endogenous and transgenichsp70 promoters in salivary glands when these promoters are induced by heat shock. We also determined that mutations introduced into the region where the polymerase pauses do not inhibit pausing in a cell-free system. Taken together, these results indicate that promoter-proximal pausing is dictated by the regulatory proteins interacting upstream from the core promoter region.

ACKNOWLEDGMENTS

This work was supported by research grant MCB-9723537 from the National Science Foundation and research grant GM47477 from NIH.

We thank Renato Paro for providing the Gal4p-expressing fly lines Mz-1087.hx and hs-GAL42-1 and John Lis for providing comments on the manuscript. We also thank Jim Alvarez and Scott Auerbach for experiments done at the early stages of this project.