Polymerase processivity and termination on Drosophila heat shock genes.
5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) limits RNA polymerase II transcription to a gene's 5'-end. Transcription of the uninduced Drosophila hsp70 gene is likewise restricted to the 5'-end, where the polymerase resides in a paused state. Furthermore, paused elongation complexes formed on the uninduced hsp70 gene and in DRB-inhibited reactions can both be restarted by Sarkosyl or high salt. These similarities prompted us to explore whether these complexes were generated by a block at the same polymerase modification step. In vivo UV cross-linking and KMnO4 hyperreactive site mapping show that while the naturally paused polymerase is restricted to the first approximately 42 base pairs of hsp70, DRB treatment of heat-induced cells allows the polymerase to transcribe past this site. Therefore, the DRB-sensitive step is probably not rate-limiting for hsp70 transcription under uninduced conditions. DRB treatment did, however, lead to the reduction of KMnO4 hyper-reactivity on hsp70 and hsp26 in a region correlating with open polymerase and/or early elongation complexes, suggesting a site for the DRB-sensitive polymerase modification step. Finally, we used the techniques of polymerase-DNA cross-linking and KMnO4 hyper-reactive site mapping to analyze the natural polymerase termination process at the 3'-end of the hsp26 gene. The data obtained are consistent with polymerases terminating at multiple sites downstream of the polyadenylation site.
Giardina
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Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
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D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D004330:Drosophila / Q000235:genetics*; D006360:Heat-Shock Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D011196:Potassium Permanganate; D012319:RNA Polymerase II / Q000378:metabolism*; D013728:Terminator Regions, Genetic; D014466:Ultraviolet Rays
D017931:DNA Primers; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D011196:Potassium Permanganate; D004004:Dichlororibofuranosylbenzimidazole; D012319:RNA Polymerase II