Analysis of the large inactive P-TEFb complex indicates that it contains one 7SK molecule, a dimer of HEXIM1 or HEXIM2, and two P-TEFb molecules containing Cdk9 phosphorylated at threonine 186

Abstract

Positive transcription elongation factor b (P-TEFb) regulates eukaryotic gene expression at the level of elongation, and is itself controlled by the reversible association of 7SK RNA and an RNA-binding protein, HEXIM1 or HEXIM2. To further understand how P-TEFb is regulated, we analyzed the stoichiometry of all the known components of the large, inactive P-TEFb complex. Mutational analyses of a putative coiled coil region in the carboxyl-terminal portion of HEXIM1 revealed that the protein is a dimer in solution and remains a dimer after binding to 7SK. Although a HEXIM1 dimer contains two potential RNA binding motifs and ultimately recruits two P-TEFb molecules, it associates with only one molecule of RNA. The first 172 nucleotides of the 330-nucleotide 7SK are sufficient to bind HEXIM1 or HEXIM2, and then recruit and inhibit P-TEFb. Deletion of the first 121 amino acids of HEXIM1 allowed it to inhibit P-TEFb partially in the absence of 7SK RNA. Mutation of a conserved tyrosine (Tyr 271 in HEXIM1) to alanine or glutamate or mutation of a conserved phenylalanine (Phe208) to alanine, aspartate, or lysine, resulted in loss of inhibition of P-TEFb, but did not affect formation of the 7SK-HEXIM-P-TEFb complex. Analysis of T-loop phosphorylation in Cdk9 indicated that phosphorylation of Thr186, but not Ser175, was essential for kinase activity and for recruitment of P-TEFb to the 7SK-HEXIM complex. A model illustrates what is currently known about how HEXIM proteins, 7SK, and P-TEFb assemble to maintain an activated kinase in a readily available, but inactive form. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.