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.
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.
Bélanger
François
F
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605-2324, USA.
Baigude
Huricha
H
Rana
Tariq M
TM
D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000528:radiation effects*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D055668:Photochemical Processes / Q000528:radiation effects; D042925:Positive Transcriptional Elongation Factor B / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000528:radiation effects; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism*
D003432:Cross-Linking Reagents; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D042925:Positive Transcriptional Elongation Factor B