Transcription-dependent association of multiple positive transcription elongation factor units to a HEXIM multimer.
The positive transcription elongation factor (P-TEFb) comprises a kinase, CDK9, and a Cyclin T1 or T2. Its activity is inhibited by association with the HEXIM1 or HEXIM2 protein bound to 7SK small nuclear RNA. HEXIM1 and HEXIM2 were found to form stable homo- and hetero-oligomers. Using yeast two-hybrid and transfection assays, we have now shown that the C-terminal domains of HEXIM proteins directly interact with each other. Hydrodynamic parameters measured by glycerol gradient ultracentrifugation and gel-permeation chromatography demonstrate that both purified recombinant and cellular HEXIM1 proteins form highly anisotropic particles. Chemical cross-links suggest that HEXIM1 proteins form dimers. The multimeric nature of HEXIM1 is maintained in P-TEFb.HEXIM1.7SK RNA complexes. Multiple P-TEFb modules are found in the inactive P-TEFb.HEXIM1.7SK complexes. It is proposed that 7SK RNA binding to a HEXIM1 multimer promotes the simultaneous recruitment and hence inactivation of multiple P-TEFb units.
Unite Mixte de Recherche 8541 CNRS, Ecole Normale Supérieure, Laboratoire de Régulation de l'Expression Génétique, 75230 Paris Cedex 05, France.
Cyprien
C
Dulac
Annemieke A
AA
Michels
Alessandro
A
Fraldi
François
F
Bonnet
Van Trung
VT
Nguyen
Giuliana
G
Napolitano
Luigi
L
Lania
Olivier
O
Bensaude
D016880:Anisotropy; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents / Q000494:pharmacology; D056906:Cyclin T; D016213:Cyclins / Q000737:chemistry; D019281:Dimerization; D005990:Glycerol / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D010957:Plasmids / Q000378:metabolism; D042925:Positive Transcriptional Elongation Factor B / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry; D014157:Transcription Factors; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques; D014461:Ultracentrifugation
C503684:CCNT1 protein, human; D003432:Cross-Linking Reagents; D056906:Cyclin T; D016213:Cyclins; C476123:HEXIM1 protein, human; C501428:HEXIM2 protein, human; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017411:Ribonucleoproteins, Small Nuclear; D014157:Transcription Factors; D012313:RNA; D042925:Positive Transcriptional Elongation Factor B; D005990:Glycerol