SCHULTE, Antje, Nadine CZUDNOCHOWSKI, Matjaz BARBORIC, Anrdé SCHONICHEN, Dalibor BLAŽEK, B Matija PETERLIN and 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, vol. 280, No 26, p. 24968-24977. ISSN 0021-9258. Available from: https://dx.doi.org/10.1074/jbc.M501431200. |
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@article{987841, author = {Schulte, Antje and Czudnochowski, Nadine and Barboric, Matjaz and Schonichen, Anrdé and Blažek, Dalibor and Peterlin, B Matija and Geyer, Matthias}, article_location = {Bethesda, USA}, article_number = {26}, doi = {http://dx.doi.org/10.1074/jbc.M501431200}, 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}, language = {eng}, issn = {0021-9258}, journal = {Journal of Biological Chemistry}, title = {Identification of a cyclin T-binding domain in Hexim1 and biochemical analysis of its binding competition with HIV-1 Tat}, url = {http://www.jbc.org/content/280/26/24968.full.pdf}, volume = {280}, year = {2005} }
TY - JOUR ID - 987841 AU - Schulte, Antje - Czudnochowski, Nadine - Barboric, Matjaz - Schonichen, Anrdé - Blažek, Dalibor - Peterlin, B Matija - Geyer, Matthias PY - 2005 TI - Identification of a cyclin T-binding domain in Hexim1 and biochemical analysis of its binding competition with HIV-1 Tat JF - Journal of Biological Chemistry VL - 280 IS - 26 SP - 24968-24977 EP - 24968-24977 PB - Amer. Soc. Biochem. Mol. Biol. SN - 00219258 KW - RNA-POLYMERASE-II KW - ELONGATION-FACTOR-B KW - IMMUNODEFICIENCY-VIRUS TRANSCRIPTION KW - DEPENDENT KINASE CDK6 KW - SMOOTH-MUSCLE-CELLS KW - P-TEFB COMPLEX KW - POSITIVE TRANSCRIPTION KW - 7SK SNRNA KW - CRYSTAL-STRUCTURE KW - GENE-EXPRESSION UR - http://www.jbc.org/content/280/26/24968.full.pdf N2 - 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. ER -
SCHULTE, Antje, Nadine CZUDNOCHOWSKI, Matjaz BARBORIC, Anrdé SCHONICHEN, Dalibor BLAŽEK, B Matija PETERLIN and Matthias GEYER. Identification of a cyclin T-binding domain in Hexim1 and biochemical analysis of its binding competition with HIV-1 Tat. \textit{Journal of Biological Chemistry}. Bethesda, USA: Amer. Soc. Biochem. Mol. Biol., 2005, vol.~280, No~26, p.~24968-24977. ISSN~0021-9258. Available from: https://dx.doi.org/10.1074/jbc.M501431200.
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