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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Motifs
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Amino Acid Sequence
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Cyclin T
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Cyclin-Dependent Kinase 9 / metabolism*
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Cyclins / metabolism*
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Electrophoretic Mobility Shift Assay
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Escherichia coli / genetics
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Glutathione Transferase / metabolism
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HeLa Cells
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Humans
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Models, Biological
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Molecular Sequence Data
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Mutation
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Positive Transcriptional Elongation Factor B / antagonists & inhibitors
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Positive Transcriptional Elongation Factor B / genetics
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Positive Transcriptional Elongation Factor B / metabolism*
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Precipitin Tests
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Protein Structure, Tertiary
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RNA, Small Nuclear / metabolism*
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RNA-Binding Proteins / chemistry
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / isolation & purification
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RNA-Binding Proteins / metabolism*
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / isolation & purification
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Recombinant Fusion Proteins / metabolism
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Transcription Factors
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Two-Hybrid System Techniques
Substances
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CCNT1 protein, human
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Cyclin T
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Cyclins
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HEXIM1 protein, human
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RNA, Small Nuclear
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RNA-Binding Proteins
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Recombinant Fusion Proteins
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Transcription Factors
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Glutathione Transferase
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Positive Transcriptional Elongation Factor B
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Cyclin-Dependent Kinase 9