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.
Mesh Terms:
Calorimetry, Chromatography, Gel, Cyclin T, Cyclins, Dimerization, Electrophoresis, Polyacrylamide Gel, Escherichia coli, Gene Products, tat, Glutathione Transferase, HeLa Cells, Humans, Kinetics, Microscopy, Fluorescence, Models, Genetic, Positive Transcriptional Elongation Factor B, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, RNA, Small Nuclear, RNA-Binding Proteins, Software, Spectrometry, Fluorescence, Thermodynamics, Time Factors, Transcription, Genetic
Calorimetry, Chromatography, Gel, Cyclin T, Cyclins, Dimerization, Electrophoresis, Polyacrylamide Gel, Escherichia coli, Gene Products, tat, Glutathione Transferase, HeLa Cells, Humans, Kinetics, Microscopy, Fluorescence, Models, Genetic, Positive Transcriptional Elongation Factor B, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, RNA, Small Nuclear, RNA-Binding Proteins, Software, Spectrometry, Fluorescence, Thermodynamics, Time Factors, Transcription, Genetic
J. Biol. Chem.
Date: Jul. 01, 2005
PubMed ID: 15855166
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