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.
Michels
Annemieke A
AA
UMR 8541 CNRS, Ecole Normale Supérieure, Régulation de l'Expression Génétique, Paris, France.
Fraldi
Alessandro
A
Li
Qintong
Q
Adamson
Todd E
TE
Bonnet
François
F
Nguyen
Van Trung
VT
Sedore
Stanley C
SC
Price
Jason P
JP
Price
David H
DH
Lania
Luigi
L
Bensaude
Olivier
O
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D056906:Cyclin T; D042863:Cyclin-Dependent Kinase 9 / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D042925:Positive Transcriptional Elongation Factor B / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D012342:RNA, Small Nuclear / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors; D020798:Two-Hybrid System Techniques
C503684:CCNT1 protein, human; D056906:Cyclin T; D016213:Cyclins; C476123:HEXIM1 protein, human; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D005982:Glutathione Transferase; D042925:Positive Transcriptional Elongation Factor B; D042863:Cyclin-Dependent Kinase 9