Publication details
Identification of a cyclin T-binding domain in Hexim1 and biochemical analysis of its binding competition with HIV-1 Tat
| Authors | |
|---|---|
| Year of publication | 2005 |
| Type | Article in Periodical |
| Magazine / Source | Journal of Biological Chemistry |
| Citation | |
| web | http://www.jbc.org/content/280/26/24968.full.pdf |
| Doi | http://dx.doi.org/10.1074/jbc.M501431200 |
| Field | Biochemistry |
| Keywords | RNA-POLYMERASE-II; ELONGATION-FACTOR-B; IMMUNODEFICIENCY-VIRUS TRANSCRIPTION; DEPENDENT KINASE CDK6; SMOOTH-MUSCLE-CELLS; P-TEFB COMPLEX; POSITIVE TRANSCRIPTION; 7SK SNRNA; CRYSTAL-STRUCTURE; GENE-EXPRESSION |
| Description | 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 mu M. 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. |