title: The role of the C-terminal helix of U1A protein in the interaction with U1hpII RNA.
abstract: Previous kinetic investigations of the N-terminal RNA Recognition Motif (RRM) domain of spliceosomal A protein of the U1 small nuclear ribonucleoprotein particle (U1A) interacting with its RNA target U1 hairpin II (U1hpII) provided experimental evidence for a 'lure and lock' model of binding. The final step of locking has been proposed to involve conformational changes in an α-helix immediately C-terminal to the RRM domain (helix C), which occludes the RNA binding surface in the unbound protein. Helix C must shift its position to accommodate RNA binding in the RNA-protein complex. This results in a new hydrophobic core, an intraprotein hydrogen bond and a quadruple stacking interaction between U1A and U1hpII. Here, we used a surface plasmon resonance-based biosensor to gain mechanistic insight into the role of helix C in mediating the interaction with U1hpII. Truncation, removal or disruption of the helix exposes the RNA-binding surface, resulting in an increase in the association rate, while simultaneously reducing the ability of the complex to lock, reflected in a loss of complex stability. Disruption of the quadruple stacking interaction has minor kinetic effects when compared with removal of the intraprotein hydrogen bonds. These data provide new insights into the mechanism whereby sequences C-terminal to an RRM can influence RNA binding.
authors.affiliation: Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Law
authors.forename: Diane S
authors.initials: DS
authors.lastname: Lee
authors.forename: Charlene S
authors.initials: CS
authors.lastname: Lee
authors.forename: Paul P
authors.initials: PP
authors.lastname: Anglim
authors.forename: Ian S
authors.initials: IS
authors.lastname: Haworth
authors.forename: Ite A
authors.initials: IA
authors.lastname: Laird-Offringa
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012342:RNA, Small Nuclear / Q000737:chemistry* / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D012342:RNA, Small Nuclear; D017412:Ribonucleoprotein, U1 Small Nuclear; C405483:U1 small nuclear RNA; C075339:U1A protein; D001224:Aspartic Acid