title: Identification of molecular contacts between the U1 A small nuclear ribonucleoprotein and U1 RNA.
abstract: We recently determined the crystal structure of the RNP domain of the U1 small nuclear ribonucleoprotein A and identified Arg and Lys residues involved in U1 RNA binding. These residues are clustered around the two highly conserved segments, RNP1 and RNP2, located in the central two beta strands. We have now studied the U1 RNA binding of mutants where potentially hydrogen bonding residues on the RNA binding surface were replaced by non-hydrogen bonding residues. In the RNP2 segment, the Thr11----Val and Asn15----Val mutations completely abolished, and the Tyr13----Phe and Asn16----Val mutations substantially reduced the U1 RNA binding, suggesting that these residues form hydrogen bonds with the RNA. In the RNP1 segment Arg52----Gln abolished, but Arg52----Lys only slightly affected U1 RNA binding, suggesting that Arg52 may form a salt bridge with phosphates of U1 RNA. Ethylation protection experiments of U1 RNA show that the backbone phosphates of the 3' two-thirds of loop II and the 5' stem are in contact with the U1 A protein. The U1 A protein-U1 RNA binding constant is substantially reduced by A----G and G----A replacements in loop II, but not by C----U or U----C replacements. Based on these biochemical data we propose a structure for the complex between the U1 A ribonucleoprotein and U1 RNA.
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: T H
authors.initials: TH
authors.lastname: Jessen
authors.forename: C
authors.initials: C
authors.lastname: Oubridge
authors.forename: C H
authors.initials: CH
authors.lastname: Teo
authors.forename: C
authors.initials: C
authors.lastname: Pritchard
authors.forename: K
authors.initials: K
authors.lastname: Nagai
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012342:RNA, Small Nuclear / Q000737:chemistry*; D012261:Ribonucleoproteins / Q000737:chemistry*; D017411:Ribonucleoproteins, Small Nuclear
chemical_list: D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear