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

Autor: Alessandro Fraldi, Stanley C. Sedore, Todd E. Adamson, Olivier Bensaude, Van Trung Nguyen, David H. Price, Luigi Lania, Qintong Li, François Bonnet, Annemieke A. Michels, Jason P. Price
Přispěvatelé: Michels, Aa, Fraldi, A, Li, D, Adamson, Te, Bonnet, F, Nguyen, Vt, Sedore, Ac, Price, Jp, Price, Dh, Lania, Luigi, Bensaude, O., 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.
Rok vydání: 2004
Předmět:
Positive Transcriptional Elongation Factor B
Recombinant Fusion Proteins
Amino Acid Motifs
Molecular Sequence Data
Electrophoretic Mobility Shift Assay
Prp24
RNA-binding protein
RNA polymerase II
RNA-Binding Protein
Biology
HeLa Cell
Models
Biological
Article
General Biochemistry
Genetics and Molecular Biology
P-TEFb
HEXIM1
Precipitin Test
Cyclins
RNA
Small Nuclear
Two-Hybrid System Techniques
7SK RNA
Escherichia coli
Humans
snRNP
Amino Acid Sequence
Molecular Biology
Glutathione Transferase
General Immunology and Microbiology
Cyclin T
General Neuroscience
RNA-Binding Proteins
MAQ1
Precipitin Tests
Cyclin-Dependent Kinase 9
Molecular biology
Cyclin
Protein Structure
Tertiary
Mutation
Amino Acid Motif
biology.protein
Transcription
Small nuclear RNA
HeLa Cells
Transcription Factors
Human
Recombinant Fusion Protein
Zdroj: The EMBO Journal. 23:2608-2619
ISSN: 1460-2075
0261-4189
DOI: 10.1038/sj.emboj.7600275
Popis: 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.
Databáze: OpenAIRE
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