Analyses of Promoter-Proximal Pausing by RNA Polymerase II on the hsp70 Heat Shock Gene Promoter in a Drosophila Nuclear Extract

Abstract

Analyses of Drosophila cells have revealed that RNA polymerase II is paused in a region 20 to 40 nucleotides downstream from the transcription start site of the hsp70 heat shock gene when the gene is not transcriptionally active. We have developed a cell-free system that reconstitutes this promoter-proximal pausing. The paused polymerase has been detected by monitoring the hyperreactivity of thymines in the transcription bubble toward potassium permanganate. The pattern of permanganate reactivity for the hsp70 promoter in the reconstituted system matches the pattern found on the promoter after it has been introduced back into flies by P-element-mediated transposition. Matching patterns of permanganate reactivity are also observed for a non-heat shock promoter, the histone H3 promoter. Further analysis of the hsp70 promoter in the reconstituted system reveals that pausing does not depend on sequence-specific interactions located immediately downstream from the pause site. Sequences upstream from the TATA box influence the recruitment of polymerase rather than the efficiency of pausing. Kinetic analysis indicates that the polymerase rapidly enters the paused state and remains stably in this state for at least 25 min. Further analysis shows that the paused polymerase will initially resume elongation when Sarkosyl is added but loses this capacity within minutes of pausing. Using an alpha-amanitin-resistant polymerase, we provide evidence that promoter-proximal pausing does not require the carboxy-terminal domain of the polymerase.