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Binding of the 7SK snRNA turns the HEXIM1 protein into a P‐TEFb (CDK9/cyclin T) inhibitor
Author(s) -
Michels Annemieke A,
Fraldi Alessandro,
Li Qintong,
Adamson Todd E,
Bonnet François,
Nguyen Van Trung,
Sedore Stanley C,
Price Jason P,
Price David H,
Lania Luigi,
Bensaude Olivier
Publication year - 2004
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1038/sj.emboj.7600275
Subject(s) - biology , small nuclear rna , p tefb , cyclin dependent kinase 9 , microbiology and biotechnology , genetics , polymerase , rna dependent rna polymerase , protein kinase a , cyclin dependent kinase 2 , gene expression , kinase , gene , promoter
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.