title: A novel combined RNA-protein interaction analysis distinguishes HIV-1 Gag protein binding sites from structural change in the viral RNA leader.
abstract: RNA-protein interactions govern many viral and host cell processes. Conventional 'footprinting' to examine RNA-protein complex formation often cannot distinguish between sites of RNA-protein interaction and sites of RNA structural remodelling. We have developed a novel technique combining photo crosslinking with RNA 2' hydroxyl reactivity ('SHAPE') that achieves rapid and hitherto unachievable resolution of both RNA structural changes and the sites of protein interaction within an RNA-protein complex. 'XL-SHAPE' was validated using well-characterized viral RNA-protein interactions: HIV-1 Tat/TAR and bacteriophage MS2 RNA/Coat Binding Protein. It was then used to map HIV-1 Gag protein interactions on 2D and 3D models of the viral RNA leader. Distinct Gag binding sites were identified on exposed RNA surfaces corresponding to regions identified by mutagenesis as important for genome packaging. This widely applicable technique has revealed a first view of the stoichiometry and structure of the initial complex formed when HIV captures its genome.
authors.affiliation: Box 157, Cambridge University Dept. of Medicine, Addenbrooke's Hospital, Hills Rd, Cambridge CB2 0QQ.
authors.forename: Julia C
authors.initials: JC
authors.lastname: Kenyon
authors.affiliation: Box 157, Cambridge University Dept. of Medicine, Addenbrooke's Hospital, Hills Rd, Cambridge CB2 0QQ.
authors.forename: Liam J
authors.initials: LJ
authors.lastname: Prestwood
authors.affiliation: Box 157, Cambridge University Dept. of Medicine, Addenbrooke's Hospital, Hills Rd, Cambridge CB2 0QQ.
authors.forename: Andrew M L
authors.initials: AM
authors.lastname: Lever
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016679:Genome, Viral / Q000235:genetics; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008967:Molecular Biology / Q000379:methods; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism
chemical_list: D020121:5' Untranslated Regions; D012367:RNA, Viral; D054301:gag Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus