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-hy...

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Bibliographic Details
Published in:The Journal of biological chemistry 2005-08, Vol.280 (34), p.30619-30629
Main Authors: Dulac, Cyprien, Michels, Annemieke A., Fraldi, Alessandro, Bonnet, François, Nguyen, Van Trung, Napolitano, Giuliana, Lania, Luigi, Bensaude, Olivier
Format: Article
Language:English
Subjects:
Anisotropy
Centrifugation, Density Gradient
Cross-Linking Reagents - pharmacology
Cyclin T
Cyclins - chemistry
Dimerization
Glycerol - pharmacology
HeLa Cells
Humans
Immunoprecipitation
Plasmids - metabolism
Positive Transcriptional Elongation Factor B - chemistry
Positive Transcriptional Elongation Factor B - metabolism
Protein Binding
Protein Structure, Tertiary
Recombinant Proteins - chemistry
Ribonucleoproteins, Small Nuclear - chemistry
RNA - chemistry
RNA-Binding Proteins - chemistry
RNA-Binding Proteins - metabolism
Transcription Factors
Transcription, Genetic
Two-Hybrid System Techniques
Ultracentrifugation
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M502471200