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

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.