Identification of a cyclin T-binding domain in Hexim1 and biochemical analysis of its binding competition with HIV-1 Tat.
The active form of the positive transcription elongation factor b (P-TEFb) consists of cyclin T and the kinase Cdk9. P-TEFb stimulates transcription by phosphorylating the C-terminal domain of RNA polymerase II. It becomes inactivated when associated in a tetrameric complex with the abundant 7SK small nuclear RNA and the recently identified protein Hexim1. In this study, we identified a stable and soluble C-terminal domain (residues 255-359) in Hexim1 of 12.5-kDa size that binds the cyclin boxes of Cyclin T1. Functional assays in HeLa cells showed that this cyclin T-binding domain (TBD) is required for the binding of Hexim1 to P-TEFb and inhibition of transcriptional activity in vivo. Analytical gel filtration and GST pull-down experiments revealed that both full-length Hexim1 and the TBD are homodimers. Isothermal titration calorimetry yielded a weak multimer for the TBD with a multimerization constant of 1.3 x 10(3) m. The binding affinity between the TBD and cyclin T1 was analyzed with fluorescence spectroscopy methods, using a dansyl-based fluorescence label at position G257C. Equilibrium fluorescence titration and stopped flow fast kinetics yield a dissociation constant of 1.2 mum. Finally, we tested the effect of the HIV-1 Tat protein on the cyclin T1-TBD complex formation. GST pull-down experiments and size exclusion chromatography exhibit a mutually exclusive binding of the two effectors to cyclin T1. Our data suggest a model where HIV-1 Tat competes with Hexim1 for cyclin T1 binding, thus releasing P-TEFb from the inactive complex to stimulate the transcription of HIV-1 gene expression.
Schulte
Antje
A
Max-Planck-Institut für Molekulare Physiologie, Abteilung Physikalische Biochemie, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany.
Czudnochowski
Nadine
N
Barboric
Matjaz
M
Schönichen
André
A
Blazek
Dalibor
D
Peterlin
B Matija
BM
Geyer
Matthias
M
D002151:Calorimetry; D002850:Chromatography, Gel; D056906:Cyclin T; D016213:Cyclins / Q000737:chemistry*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015696:Gene Products, tat / Q000737:chemistry*; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D042925:Positive Transcriptional Elongation Factor B / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012342:RNA, Small Nuclear / Q000737:chemistry; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D012984:Software; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013997:Time Factors; D014157:Transcription Factors; D014158:Transcription, Genetic
C503684:CCNT1 protein, human; D056906:Cyclin T; D016213:Cyclins; D015696:Gene Products, tat; C476123:HEXIM1 protein, human; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D005982:Glutathione Transferase; D042925:Positive Transcriptional Elongation Factor B