Relief of two built-In autoinhibitory mechanisms in P-TEFb is required for assembly of a multicomponent transcription elongation complex at the human immunodeficiency virus type 1 promoter.
Tat stimulation of human immunodeficiency virus type 1 (HIV-1) transcription requires Tat-dependent recruitment of human positive transcription elongation factor b (P-TEFb) to the HIV-1 promoter and the formation on the trans-acting response element (TAR) RNA of a P-TEFb-Tat-TAR ternary complex. We show here that the P-TEFb heterodimer of Cdk9-cyclin T1 is intrinsically incapable of forming a stable complex with Tat and TAR due to two built-in autoinhibitory mechanisms in P-TEFb. Both mechanisms exert little effect on the P-TEFb-Tat interaction but prevent the P-TEFb-Tat complex from binding to TAR RNA. The first autoinhibition arises from the unphosphorylated state of Cdk9, which establishes a P-TEFb conformation unfavorable for TAR recognition. Autophosphorylation of Cdk9 overcomes this inhibition by inducing conformational changes in P-TEFb, thereby exposing a region in cyclin T1 for possible TAR binding. An intramolecular interaction between the N- and C-terminal regions of cyclin T1 sterically blocks the P-TEFb-TAR interaction and constitutes the second autoinhibitory mechanism. This inhibition is relieved by the binding of the C-terminal region of cyclin T1 to the transcription elongation factor Tat-SF1 and perhaps other cellular factors. Upon release from the intramolecular interaction, the C-terminal region also interacts with RNA polymerase II and is required for HIV-1 transcription, suggesting its role in bridging the P-TEFb-Tat-TAR complex and the basal elongation apparatus. These data reveal novel control mechanisms for the assembly of a multicomponent transcription elongation complex at the HIV-1 promoter.
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720-3206, USA.
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D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000235:genetics; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D010766:Phosphorylation; D042925:Positive Transcriptional Elongation Factor B; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014779:Virus Replication / Q000235:genetics; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D054322:tat Gene Products, Human Immunodeficiency Virus; D042925:Positive Transcriptional Elongation Factor B; D017346:Protein Serine-Threonine Kinases; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases