Premium
Purification of a Tat‐associated kinase reveals a TFIIH complex that modulates HIV‐1 transcription
Author(s) -
GarcíaMartínez León F.,
Mavankal Gopinath,
Neveu John M.,
Lane William S.,
Ivanov Dmitri,
Gaynor Richard B.
Publication year - 1997
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/16.10.2836
Subject(s) - biology , transcription factor ii h , transcription (linguistics) , microbiology and biotechnology , virology , kinase , genetics , gene expression , promoter , gene , linguistics , philosophy
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 with 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 II 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.