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 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.
García-Martínez
L F
LF
Department of Medicine, University of Texas Southwestern Medical Center, Dallas 75235-8594, USA.
Mavankal
G
G
Neveu
J M
JM
Lane
W S
WS
Ivanov
D
D
Gaynor
R B
RB
D018844:Cyclin-Dependent Kinases; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics*; D009363:Neoplasm Proteins / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D042925:Positive Transcriptional Elongation Factor B; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism*; D017421:Sequence Analysis; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D019473:Transcription Factors, TFII; D014158:Transcription, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D015696:Gene Products, tat; D009363:Neoplasm Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D054322:tat Gene Products, Human Immunodeficiency Virus; D051758:Transcription Factor TFIIH; D004004:Dichlororibofuranosylbenzimidazole; D042925:Positive Transcriptional Elongation Factor B; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases; D012319:RNA Polymerase II; D000095043:Cyclin-Dependent Kinase-Activating Kinase