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

J 2005

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 et. al.

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

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

Odkazy

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

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 23. 7. 2012 06:27, Olga Křížová

Anotace

V originále

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.

Citovat

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.

@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 a 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, roč.~280, č.~26, s.~24968-24977. ISSN~0021-9258. Dostupné z: https://dx.doi.org/10.1074/jbc.M501431200.

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