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

Details

Serval ID
serval:BIB_C61A22494742
Type
Article: article from journal or magazin.
Collection
Publications
Title
Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor.
Journal
Embo Journal
Author(s)
Michels A.A., Fraldi A., Li Q., Adamson T.E., Bonnet F., Nguyen V.T., Sedore S.C., Price J.P., Price D.H., Lania L., Bensaude O.
ISSN
0261-4189 (Print)
ISSN-L
0261-4189
Publication state
Published
Issued date
2004
Peer-reviewed
Oui
Volume
23
Number
13
Pages
2608-2619
Language
english
Notes
Publication types: Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, P.H.S.
Publication Status: ppublish
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.
Keywords
Amino Acid Motifs, Amino Acid Sequence, Cyclin T, Cyclin-Dependent Kinase 9/metabolism, Cyclins/metabolism, Electrophoretic Mobility Shift Assay, Escherichia coli/genetics, Glutathione Transferase/metabolism, HeLa Cells, Humans, Models, Biological, Molecular Sequence Data, Mutation, Positive Transcriptional Elongation Factor B/antagonists & inhibitors, Positive Transcriptional Elongation Factor B/genetics, Precipitin Tests, Protein Structure, Tertiary, RNA, Small Nuclear/metabolism, RNA-Binding Proteins/chemistry, RNA-Binding Proteins/genetics, Recombinant Fusion Proteins/chemistry, Recombinant Fusion Proteins/isolation & purification, Two-Hybrid System Techniques
Pubmed
Web of science
Create date
28/07/2008 16:00
Last modification date
20/08/2019 15:41
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