Direct evidence that HIV-1 Tat stimulates RNA polymerase II carboxyl-terminal domain hyperphosphorylation during transcriptional elongation.
The human immunodeficiency virus type-1 (HIV-1) Tat protein regulates transcription by stimulating RNA polymerase processivity. Using immobilised templates, we have been able to study the effects of Tat on protein kinase activity during the pre-initiation and elongation stages of HIV-1 transcription. In pre-initiation complexes formed at the HIV-1 LTR, the C-terminal domain (CTD) of RNA polymerase II is rapidly phosphorylated by transcription factor IIH (TFIIH). Addition of Tat does not affect either the rate or the extent of CTD phosphorylation in the pre-initiation complexes. By contrast, Tat is able to stimulate additional CTD phosphorylation in elongation complexes. This reaction creates a novel form of the RNA polymerase that we have called RNA polymerase IIo*. Formation of the RNA polymerase IIo* occurs only after transcription of templates carrying a functional TAR RNA element and is strongly inhibited by low concentrations of 5,6-dichloro-1-beta- D -ribofuranosyl benzimidazole (DRB), a potent inhibitor of CDK9, the protein kinase subunit of the Tat-associated kinase (TAK). Immunoblotting experiments have shown that CDK9 and its associated cyclin, cyclin T1, are present at equivalent levels in both the pre-initiation and elongation complexes. We conclude that activation of the CDK9 kinase, leading to CTD phosphorylation, occurs only in elongation complexes that have transcribed through the Tat-recognition element, TAR RNA.
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Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK.
Karn
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D056906:Cyclin T; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D015696:Gene Products, tat / Q000378:metabolism*; D006678:HIV / Q000235:genetics*; D016325:HIV Long Terminal Repeat / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D009687:Nuclear Proteins; D010766:Phosphorylation / Q000187:drug effects; D042925:Positive Transcriptional Elongation Factor B; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D012319:RNA Polymerase II / Q000737:chemistry* / Q000378:metabolism*; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000378:metabolism; D019473:Transcription Factors, TFII; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics*; D054322:tat Gene Products, Human Immunodeficiency Virus; D000095043:Cyclin-Dependent Kinase-Activating Kinase
C503684:CCNT1 protein, human; D056906:Cyclin T; D016213:Cyclins; D004268:DNA-Binding Proteins; D003838:Deoxyadenine Nucleotides; D004791:Enzyme Inhibitors; D015696:Gene Products, tat; D007546:Isoquinolines; D009687:Nuclear Proteins; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D054322:tat Gene Products, Human Immunodeficiency Virus; D051758:Transcription Factor TFIIH; D004004:Dichlororibofuranosylbenzimidazole; C053177:N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide; D000255:Adenosine Triphosphate; D042925:Positive Transcriptional Elongation Factor B; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases; D012319:RNA Polymerase II; D000469:Alkaline Phosphatase; C026600:2'-deoxyadenosine triphosphate; D000095043:Cyclin-Dependent Kinase-Activating Kinase