An evolutionary conserved Hexim1 peptide binds to the Cdk9 catalytic site to inhibit P-TEFb

The positive transcription elongation factor (P-TEFb) is required for transcription of most genes by RNA polymerase II. Hexim proteins associated to 7SK, a noncoding RNA, bind to P-TEFb and reversibly inhibit its activity. This transcription factor comprises the kinase Cdk9 and a cyclin T. Using a photoreactive amino acid incorporated into proteins, we provide the first evidence of a direct interaction between Cdk9 and Hexim1, in live cells, in cell extracts and in in vitro assembled complexes. An evolutionary conserved Hexim1 peptide, the "PYNT" sequence, cross-links to the Cdk9 activation segment that controls access to the catalytic cleft. Interference with binding of substrates accounts for kinase inhibition.