Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor

Annemieke A Michels; Alessandro Fraldi; Qintong Li; Todd E Adamson; François Bonnet; Van Trung Nguyen; Stanley C Sedore; Jason P Price; David H Price; Luigi Lania; Olivier Bensaude

doi:10.1038/sj.emboj.7600275

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Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor

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Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor

Annemieke A Michels, Alessandro Fraldi, Qintong Li, Todd E Adamson, François Bonnet, Van Trung Nguyen, Stanley C Sedore, Jason P Price, David H Price, Luigi Lania, …

The EMBO journal, Vol.23(13), pp.2608-2619

07/07/2004

DOI: 10.1038/sj.emboj.7600275

PMCID: PMC449783

PMID: 15201869

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Abstract

The positive transcription elongation factor b (P-TEFb) plays a pivotal role in productive elongation of nascent RNA molecules by RNA polymerase II. Core active P-TEFb is composed of CDK9 and cyclin T. In addition, mammalian cell extracts contain an inactive P-TEFb complex composed of four components, CDK9, cyclin T, the 7SK snRNA and the MAQ1/HEXIM1 protein. We now report an in vitro reconstitution of 7SK-dependent HEXIM1 association to purified P-TEFb and subsequent CDK9 inhibition. Yeast three-hybrid tests and gel-shift assays indicated that HEXIM1 binds 7SK snRNA directly and a 7SK snRNA-recognition motif was identified in the central part of HEXIM1 (amino acids (aa) 152–155). Data from yeast two-hybrid and pull-down assay on GST fusion proteins converge to a direct binding of P-TEFb to the HEXIM1 C-terminal domain (aa 181–359). Consistently, point mutations in an evolutionarily conserved motif (aa 202–205) were found to suppress P-TEFb binding and inhibition without affecting 7SK recognition. We propose that the RNA-binding domain of HEXIM1 mediates its association with 7SK and that P-TEFb then enters the complex through association with HEXIM1.

HEXIM1

transcription

RNA polymerase II

MAQ1

P-TEFb

Details

Title: Subtitle: Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor
Creators: Annemieke A Michels - UMR 8541 CNRS, Ecole Normale Supérieure, Régulation de l'Expression Génétique, Paris, France
Alessandro Fraldi - Dipartimento di Genetica, Biologia Generale e Molecolare, Università di Napoli ‘Federico II', Napoli, Italy
Qintong Li - Department of Biochemistry, University of Iowa, Iowa City, IA, USA
Todd E Adamson - Department of Biochemistry, University of Iowa, Iowa City, IA, USA
François Bonnet - UMR 8541 CNRS, Ecole Normale Supérieure, Régulation de l'Expression Génétique, Paris, France
Van Trung Nguyen - UMR 8541 CNRS, Ecole Normale Supérieure, Régulation de l'Expression Génétique, Paris, France
Stanley C Sedore - Medical Scientist Training Program, University of Iowa, Iowa City, IA, USA
Jason P Price - Department of Biochemistry, University of Iowa, Iowa City, IA, USA
David H Price - Department of Biochemistry, University of Iowa, Iowa City, IA, USA
Luigi Lania - Dipartimento di Genetica, Biologia Generale e Molecolare, Università di Napoli ‘Federico II', Napoli, Italy
Olivier Bensaude - UMR 8541 CNRS, Ecole Normale Supérieure, Régulation de l'Expression Génétique, Paris, France
Resource Type: Journal article
Publication Details: The EMBO journal, Vol.23(13), pp.2608-2619
DOI: 10.1038/sj.emboj.7600275
PMID: 15201869
PMCID: PMC449783
NLM abbreviation: EMBO J
ISSN: 0261-4189
eISSN: 1460-2075
Publisher: Nature Publishing Group
Alternative title: 7SK/HEXIM1-mediated inhibition of P-TEFb
Language: English
Date published: 07/07/2004
Academic Unit: Biochemistry and Molecular Biology
Record Identifier: 9984025266202771

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