PHOSPHORYLATION OF THE RNA-POLYMERASE-II LARGEST SUBUNIT DURING HEAT-SHOCK AND INHIBITION OF TRANSCRIPTION IN HELA-CELLS

The phosphorylation of the C-terminal domain (CTD) of the largest subu nit of eukaryotic RNA polymerase II has been investigated in HeLa cell s exposed to heat shock, in control cells, the phosphorylated subunit, Ilo, and the dephosphorylated subunit, lla, were round in similar amo unts. During heat shock, however, the phosphorylated subunit, Ilo, acc umulated, whereas the amount of Ila subunit decreased. Since phosphory lation of the CTD had been suggested to play a role in the initiation of transcription and since heat shock was known to perturb gene expres sion at the level of transcription, the phosphorylation state of RNA p olymerase II was examined in cells that had been treated with various inhibitors of transcription. Under normal growth temperature, actinomy cin D (over 0.1 mu g/ml) and okadaic acid, a phosphatase inhibitor, we re found to inhibit polymerase dephosphorylation. Whereas 5,6-dichloro benzimidazole riboside (DRB), N-(2-[Methylamino]ethyl)-5-isoquinolines ulfon (H-8), and actinomycin D (over 5 mu g/ml) were found to inhibit polymerase phosphorylation. Actinomycin D concentrations, which inhibi ted the dephosphorylation process, were lower than those required to i nhibit the phosphorylation process. In contrast, during heat shock or exposure to sodium arsenite, a chemical inducer of the heat-shock resp onse, the phosphorylated subunit, Ilo, accumulated even in the presenc e of inhibitors of transcription such as DRB, H-8, and actinomycin D. These experiments demonstrated the existence of a heat-shock-induced C TD-phosphorylation process that might contribute to the regulation of transcription during stress.(C) 1994 Wiley-Liss, Inc.