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  • Title: U30 of 7SK RNA forms a specific photo-cross-link with Hexim1 in the context of both a minimal RNA-binding site and a fully reconstituted 7SK/Hexim1/P-TEFb ribonucleoprotein complex.
    Author: Bélanger F, Baigude H, Rana TM.
    Journal: J Mol Biol; 2009 Mar 06; 386(4):1094-107. PubMed ID: 19244621.
    Abstract:
    Eukaryotic transcription by RNA polymerase II is a highly regulated process and divided into three major steps: initiation, elongation, and termination. Each step of transcription is controlled by a number of cellular factors. Positive transcription factor b, P-TEFb, is composed of cyclin-dependent kinase 9 and a regulatory cyclin (T1/T2). P-TEFb promotes transcriptional elongation of RNA polymerase II by using the catalytic function of CDK9 to phosphorylate various substrates during transcription. P-TEFb is inactivated by sequestration in a complex with the Hexim1 protein and 7SK RNA. The structure of this inactive P-TEFb complex and the mechanisms controlling its equilibrium with the active complex are poorly understood. Here, we used a photoactive nucleotide, 4-thioU, to study the interactions between 7SK RNA and Hexim1. We identified a specific cross-link between nucleotide U30 of 7SK RNA and amino acids 210-220 of Hexim1, in the context of both a minimal RNA-binding site and a fully reconstituted 7SK/Hexim1/P-TEFb ribonucleoprotein complex. We show also that a minimal 7SK RNA hairpin comprising nucleotides 24-87 can bind specifically to Hexim1 in vivo. Our results demonstrate directly that the Hexim1 binding site is located in the 24-87 region of 7SK RNA and that the protein residues outside the basic domain of Hexim1 are involved in specific RNA interactions.
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