Transcription elongation factor P-TEFb mediates Tat activation of HIV-1 transcription at multiple stages.
Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcription elongation through recognition of the transactivation response (TAR) RNA stem-loop structure at the 5' end of nascent viral transcripts. Recently, a human transcription elongation factor P-TEFb, consisting of CDK9 kinase, cyclin T and other associated factors, has been shown to interact with Tat to restore Tat activation in HeLa nuclear extract depleted of P-TEFb. Here, we report the purification of a P-TEFb complex fraction containing epitope-tagged wild-type CDK9 or kinase-inactive CDK9 and five tightly associated polypeptides. Only wild-type P-TEFb complex with an active CDK9 kinase was able to hyperphosphorylate the C-terminal domain of RNA polymerase II and mediate Tat transactivation in P-TEFb-depleted HeLa nuclear extract. Tat also stimulated transcription elongation by recruitment of the P-TEFb complex to the HIV-1 promoter through a Tat-TAR interaction. A possible mechanism for P-TEFb to become associated with polymerase elongation complexes and function as a general elongation factor was demonstrated by an interaction of P-TEFb with double-stranded RNA molecules through an 87 kDa subunit. Finally, P-TEFb was found to interact with and phosphorylate Tat-SF1, a Tat cofactor required for Tat transactivation. Our data indicate that the various subunits of the human P-TEFb complex may play distinct roles at multiple stages to mediate Tat activation of HIV-1 transcription elongation.
Zhou
Q
Q
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA. qzhou@uclink4.berkeley.edu
Chen
D
D
Pierstorff
E
E
Luo
K
K
D001483:Base Sequence; D002461:Cell Line, Transformed; D016213:Cyclins / Q000378:metabolism; D015696:Gene Products, tat / Q000235:genetics*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010441:Peptide Chain Elongation, Translational; D010766:Phosphorylation; D042925:Positive Transcriptional Elongation Factor B; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D015533:Transcriptional Activation; D054322:tat Gene Products, Human Immunodeficiency Virus
D016213:Cyclins; D015696:Gene Products, tat; C101819:HTATSF1 protein, human; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D015534:Trans-Activators; D054322:tat Gene Products, Human Immunodeficiency Virus; D011494:Protein Kinases; D042925:Positive Transcriptional Elongation Factor B; D017346:Protein Serine-Threonine Kinases