Identification of a Cyclin T-Binding Domain in Hexim1 and Biochemical Analysis of Its Binding Competition with HIV-1 Tat

Antje Schulte, Nadine Czudnochowski, Matjaz Barboric, André Schönichen, Dalibor Blazek, B. Matija Peterlin, Matthias Geyer
2005 Journal of Biological Chemistry  
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
more » ... inds 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 ؋ 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 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. The positive transcription elongation factor b (P-TEFb) 1 is a key regulator of the productive transcription elongation of nascent pre-mRNA molecules into mature mRNA by RNA polymerase II (1). The action of P-TEFb has been studied most
doi:10.1074/jbc.m501431200 pmid:15855166 fatcat:imreamvlk5h6xgxirezxwhg6ba