, and full-length CycT1 (amino acids 728) [CycT1(1-728)], but not truncated CycT1(1-303), was also phosphorylated by CDK9. P-TEFb complexes containing a catalytically inactive CDK9 mutant (D167N) bound TAR RNA weakly and independently of ATP, as did a C-terminal truncated CDK9 mutant that was catalytically active but unable to undergo autophosphorylation. Analysis of different Tat proteins revealed that the 101-amino-acid SF2 HIV-1 Tat was unable to bind TAR with CycT1(1-303) in the absence of phosphorylated CDK9, whereas unphosphorylated CDK9 strongly blocked binding of HIV-2 Tat to TAR RNA in a manner that was reversed upon autophosphorylation. Replacement of CDK9 phosphorylation sites with negatively charged residues restored binding of CycT1(1-303)-D167N-Tat, and rendered D167N a more potent inhibitor of transcription in vitro. Taken together, these results demonstrate that CDK9 phosphorylation is required for high-affinity binding of Tat-P-TEFb to TAR RNA and that the state of P-TEFb phosphorylation may regulate Tat transactivation in vivo.Activation of human immunodeficiency virus type-1 (HIV-1) transcription by the virus-encoded transcription factor, Tat, provides an important paradigm for understanding the mechanisms that regulate transcription elongation by RNA polymerase II (RNAPII). Transcription complexes that form at the HIV-1 promoter in the absence of Tat are competent to initiate transcription but elongate inefficiently, due to the effects of negative general elongation factors (22,50,51,55,57; reviewed in references 16 and 56) and an inhibitory RNA structure that induces pausing of RNAPII complexes (38). Tat functions as a promoter-specific transcription elongation factor through binding to the transactivation response element (TAR) in the 5Ј-untranslated leader of viral transcripts to stimulate processive transcription by RNAPII (for a review, see references 29 and 30).Tat regulates an early step in transcription elongation that requires cyclin T1 (CycT1) and CDK9 (21,35,40,52,(58)(59)(60), which are subunits of the positive transcription elongation factor P-TEFb (36) and Tat-associated kinase (21, 23, 24) complexes. CDK9 is a Cdc2-related kinase (20) that promotes general elongation of transcription at many promoters in vitro and can phosphorylate the C-terminal domain (CTD) of the largest subunit of RNAPII (9,35,60). We previously cloned CycT1 as a protein that interacts strongly with the 48-amino-

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