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

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.

Pubmed ID: 15201869

Authors

  • Michels AA
  • Fraldi A
  • Li Q
  • Adamson TE
  • Bonnet F
  • Nguyen VT
  • Sedore SC
  • Price JP
  • Price DH
  • Lania L
  • Bensaude O

Journal

The EMBO journal

Publication Data

July 7, 2004

Associated Grants

  • Agency: NIGMS NIH HHS, Id: GM35500

Mesh Terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Cyclin T
  • Cyclin-Dependent Kinase 9
  • Cyclins
  • Electrophoretic Mobility Shift Assay
  • Escherichia coli
  • Glutathione Transferase
  • HeLa Cells
  • Humans
  • Models, Biological
  • Molecular Sequence Data
  • Mutation
  • Positive Transcriptional Elongation Factor B
  • Precipitin Tests
  • Protein Structure, Tertiary
  • RNA, Small Nuclear
  • RNA-Binding Proteins
  • Recombinant Fusion Proteins
  • Two-Hybrid System Techniques