HEAT-SHOCK INACTIVATION OF THE TFIIH-ASSOCIATED KINASE AND CHANGE IN THE PHOSPHORYLATION SITES ON THE C-TERMINAL DOMAIN OF RNA-POLYMERASE-II

The C-terminal domain (CTD) of the RNA polymerase II largest subunit ( RPB1) plays a central role in transcription. The CTD is unphosphorylat ed when the polymerase assembles into a preinitiation complex of trans cription and becomes heavily phosphorylated during promoter clearance and entry into elongation of transcription. A kinase associated to the general transcription factor TFIIH, in the preinitiation complex, pho sphorylates the CTD. The TFIIH-associated CTD kinase activity was foun d to decrease in extracts from heat-shocked HeLa cells compared to uns tressed cells. This loss of activity correlated with a decreased solub ility of the TFIIH factor. The TFIIH-kinase impairment during heat-sho ck was accompanied by the disappearance of a particular phosphoepitope (CC-3) on the RPB1 subunit. The CC-3 epitope was localized on the C-t erminal end of the CTD and generated in vitro when the RPB1 subunit wa s phosphorylated by the TFIIH-associated kinase but not by another CTD kinase such as MAP kinase. In apparent discrepancy, the overall RPB1 subunit phosphorylation increased during heat-shock. The decreased act ivity in vivo of the TFIIH kinase might be compensated by a stress-act ivated CTD kinase such as MAP kinase. These results also suggest that heat-shock gene transcription may have a weak requirement for TFIIH ki nase activity.