Identification of a cyclin T-binding domain in Hexim1 and biochemical analysis of its binding competition with HIV-1 Tat

SCHULTE, Antje, Nadine CZUDNOCHOWSKI, Matjaz BARBORIC, Anrdé SCHONICHEN, Dalibor BLAŽEK, B Matija PETERLIN a Matthias GEYER. Identification of a cyclin T-binding domain in Hexim1 and biochemical analysis of its binding competition with HIV-1 Tat. Journal of Biological Chemistry. Bethesda, USA: Amer. Soc. Biochem. Mol. Biol., 2005, roč. 280, č. 26, s. 24968-24977. ISSN 0021-9258. Dostupné z: https://dx.doi.org/10.1074/jbc.M501431200.

Základní údaje

• Originální název: Identification of a cyclin T-binding domain in Hexim1 and biochemical analysis of its binding competition with HIV-1 Tat
• Autoři: SCHULTE, Antje, Nadine CZUDNOCHOWSKI, Matjaz BARBORIC, Anrdé SCHONICHEN, Dalibor BLAŽEK, B Matija PETERLIN a Matthias GEYER.
• Vydání: Journal of Biological Chemistry, Bethesda, USA, Amer. Soc. Biochem. Mol. Biol. 2005, 0021-9258.

Další údaje

• Originální jazyk: angličtina
• Typ výsledku: Článek v odborném periodiku
• Obor: 10600 1.6 Biological sciences
• Stát vydavatele: Spojené státy
• Utajení: není předmětem státního či obchodního tajemství
• WWW: URL
• Impakt faktor: Impact factor: 5.854
• Doi: http://dx.doi.org/10.1074/jbc.M501431200
• UT WoS: 000230114000081
• Klíčová slova anglicky: 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
• Štítky: ok
• Příznaky: Mezinárodní význam, Recenzováno

Anotace

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.