Polymerase processivity and termination on Drosophila heat shock genes

• Published in: The Journal of biological chemistry Vol. 268; no. 32; pp. 23806 - 23811
• Main Authors: GIARDINA, C, LIS, J. T
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15.11.1993
• Subjects: Animals; Base Sequence; Biological and medical sciences; Cells, Cultured; Dichlororibofuranosylbenzimidazole - pharmacology; DNA Primers; DNA-Binding Proteins - metabolism; Drosophila; Drosophila - genetics; drosophila melanogaster; estres termico; Fundamental and applied biological sciences. Psychology; gene; genes; heat stress; Heat-Shock Proteins - genetics; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Potassium Permanganate; proteinas; proteine; proteins; RNA Polymerase II - metabolism; stress thermique; Terminator Regions, Genetic; Transcription. Transcription factor. Splicing. Rna processing; transferasas; transferase; transferases; Ultraviolet Rays; Enzyme; Insecta; Transferases; Drosophila; DNA-directed RNA polymerase; Drosophilidae; Transcription termination; Site specificity; Nucleotidyltransferases; Regulation(control); Enzymatic activity; Arthropoda; Heat shock protein; Invertebrata; Southern blotting; Diptera
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(20)80456-6

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 hyper-reactive 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.