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.
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 (Phe(208)) 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 Thr(186), but not Ser(175), 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.
Li
Qintong
Q
Department of Biochemistry and Molecular Biology Program, University of Iowa, Iowa City, Iowa 52242, USA.
Price
Jason P
JP
Byers
Sarah A
SA
Cheng
Dongmei
D
Peng
Junmin
J
Price
David H
DH
D000595:Amino Acid Sequence; D042846:CDC2-CDC28 Kinases / Q000737:chemistry / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances / Q000378:metabolism; D008958:Models, Molecular; D010766:Phosphorylation; D042925:Positive Transcriptional Elongation Factor B / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry / Q000378:metabolism*; D013912:Threonine; D014157:Transcription Factors
C476123:HEXIM1 protein, human; C501428:HEXIM2 protein, human; D046911:Macromolecular Substances; D016601:RNA-Binding Proteins; D017411:Ribonucleoproteins, Small Nuclear; D014157:Transcription Factors; D013912:Threonine; D042925:Positive Transcriptional Elongation Factor B; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2