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Papers In Press, published online ahead of print June 17, 2005
Department of Biochemistry, University of Iowa, Iowa City, IA 52242
Corresponding Author: david-price{at}uiowa.edu
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 (Y271 in HEXIM1) to alanine or glutamate or mutation of a conserved phenylalanine (F208) 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 T186, but not S175, 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.
J. Biol. Chem, 10.1074/jbc.M502712200
Submitted on March 11, 2005
Revised on May 23, 2005
Accepted on June 15, 2005
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
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