Purification of a Tat-associated kinase reveals a TFIIH complex that modulates HIV-1 transcription

The Tat protein is a transcriptional activator which is required for efficient human immunodeficiency virus 1 (HIV-1) gene expression Tat stimulates HIV-1 transcriptional elongation by increasing the processivity of RNA polymerase II. To address whether Tat-mediated effects on HIV-1 gene expression are due to modulation in the phosphorylation of the RNA polymerase II C-terminal domain (CTD), we developed a purification protocol to identify cellular kinases that are capable of binding to Tat and hyperphosphorylating the RNA polymerase II CTD, A 600 kDa protein complex with these properties was isolated, and specific components were identified using peptide microsequence analysis, This analysis indicated that proteins comprising the multi-subunit TFIIH complex, in addition to several novel factors, were associated,vith Tat using both in vitro and in vivo analysis, The Tat-associated kinase bound to the activation domain of Tat, and its ability to hyperphosphorylate RNA polymerase TI was markedly stimulated by Tat, Furthermore, the addition of the Tat-associated kinase to in vitro transcription assays stimulated the ability of Tat to activate HIV-1 transcription. These results define a cellular kinase complex whose activity is modulated by Tat to result in activation of HIV-1 trancription.