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J. Biol. Chem., Vol. 280, Issue 31, 28819-28826, August 5, 2005

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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^*

Qintong Li, Jason P. Price, Sarah A. Byers, Dongmei Cheng¶, Junmin Peng¶, and David H. Price||

From the Department of Biochemistry and Molecular Biology Program, University of Iowa, Iowa City, Iowa 52242 and the ^¶Department of Human Genetics, Center for Neurodegenerative Disease, Emory School of Medicine, Atlanta, Georgia 30322

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 (Tyr²⁷¹ 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.

Received for publication, March 11, 2005 , and in revised form, May 23, 2005.

^* This work was supported by National Institutes of Health Grants GM35500 and AI54340 (to D. H. P.) and American Heart Foundation Fellowship 0510040Z (to Q. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Dept. of Biochemistry, University of Iowa, Iowa City, IA 52242. Tel.: 319-335-7910; Fax: 319-384-4770; E-mail: david-price{at}uiowa.edu.

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