Mf. Dubois et al., PHOSPHORYLATION OF THE RNA-POLYMERASE-II LARGEST SUBUNIT DURING HEAT-SHOCK AND INHIBITION OF TRANSCRIPTION IN HELA-CELLS, Journal of cellular physiology, 158(3), 1994, pp. 417-426
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.