HEAT-SHOCK-SPECIFIC PHOSPHORYLATION AND TRANSCRIPTIONAL ACTIVITY OF RNA-POLYMERASE-II

The carboxyl-terminal domain (CTD) of the largest RNA polymerase II (p ol II) subunit is a target for extensive phosphorylation in vivo. Usin g in vitro kinase assays it was found that several different protein k inases can phosphorylate the CTD including the transcription factor II H-associated CDK-activating CDK7 kinase (R. Roy, J. P. Adamczewski, T. Seroz, W. Vermeulen, J. P. Tassan, L. Schaeffer, E. A. Nigg, J. H. Ho eijmakers, and J. NI. Egly, 1994, Cell 79, 1093-1101). Here we report the colocalization of CDK7 and the phosphorylated form of CTD (phospho CTD) to actively transcribing genes in intact salivary gland cells of Chironomus tentans. Following a heat-shock treatment, both CDK7 and po l II staining disappear from non-heat-shock genes concomitantly with t he abolishment of transcriptional activity of these genes. In contrast , the actively transcribing heat-shock genes, manifested as chromosoma l puff 5C on chromosome TV (IV-5C), stain intensely for phosphoCTD, bu t are devoid of CDK7. Furthermore, the staining of puff TV-BC with ant i-PCTD antibodies was not detectably influenced by the TFIIH kinase an d transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimida zole (DRB). Following heat-shock treatment, the transcription of non-h eat-shock genes was completely eliminated, while newly formed heat-sho ck gene transcripts emerged in a DRB-resistant manner. Thus, heat shoc k in these cells induces a rapid clearance of CDK7 from the non-heat-s hock genes, indicating a lack of involvement of CDK7 in the induction and function of the heat-induced genes. The results taken together sug gest the existence of heat-shock-specific CTD phosphorylation in livin g cells. This phosphorylation is resistant to DRB treatment, suggestin g that not only phosphorylation but also transcription of heat-shock g enes is DRB resistant and that CDK7 in heat shock cells is not associa ted with TFIIH. (C) 1998 Academic Press.