Tat modifies the activity of CDK9 to phosphorylate serine 5 of the RNA polymerase II carboxyl-terminal domain during human immunodeficiency virus type 1 transcription

Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcriptional elongation by recruitment of carboxyl-terminal domain (CTD) kinases to the HIV-1 promoter. Using an immobilized DNA template assay, we have analyzed t he effect of Tat on kinase activity during the initiation and elongation ph ases of HIV-1 transcription. Our results demonstrate that cyclin-dependent kinase 7 (CDK7) (TFIIH) and CDK9 (P-TEFb) both associate with the HIV-1 pre initiation complex. Hyperphosphorylation of the RNA polymerase II (RNAP II) CTD in the HIV-1 preinitiation complex, in the absence of Tat, takes place at CTD serine 2 and serine 5. Analysis of preinitiation complexes formed i n immunodepleted extracts suggests that CDK9 phosphorylates serine 2, while CDK7 phosphorylates serine 5. Remarkably, in the presence of Tat, the subs trate specificity of CDK9 is altered, such that the kinase phosphorylates b oth serine 2 and serine 5. Tat-induced CTD phosphorylation by CDK9 is stron gly inhibited by low concentrations of 5,6-dichloro-1-beta-D-ribofuranosylb enzimidazole, an inhibitor of transcription elongation by RNAP II. Analysis of stalled transcription elongation complexes demonstrates that CDK7 is re leased from the transcription complex between positions +14 and +36, prior to the synthesis of transactivation response (TAR) RNA. In contrast, CDK9 s tays associated with the complex through +79. Analysis of CTD phosphorylati on indicates a biphasic modification pattern, one in the preinitiation comp lex and the other between +36 and +79. The second phase of CTD phosphorylat ion is Tat-dependent and TAR-dependent. These studies suggest that the abil ity of Tat to increase transcriptional elongation may be due to its ability to modify the substrate specificity of the CDK9 complex.