title: Investigating the structural basis of purine specificity in the structures of MS2 coat protein RNA translational operator hairpins.
abstract: We have determined the structures of complexes between the phage MS2 coat protein and variants of the replicase translational operator in order to explore the sequence specificity of the RNA-protein interaction. The 19-nt RNA hairpins studied have substitutions at two positions that have been shown to be important for specific binding. At one of these positions, -10, which is a bulged adenosine (A) in the stem of the wild-type operator hairpin, substitutions were made with guanosine (G), cytidine (C) and two non-native bases, 2-aminopurine (2AP) and inosine (I). At the other position, -7 in the hairpin loop, the native adenine was substituted with a cytidine. Of these, only the G-10, C-10 and C-7 variants showed interpretable density for the RNA hairpin. In spite of large differences in binding affinities, the structures of the variant complexes are very similar to the wild-type operator complex. For G-10 substitutions in hairpin variants that can form bulges at alternative places in the stem, the binding affinity is low and a partly disordered conformation is seen in the electron density maps. The affinity is similar to that of wild-type when the base pairs adjacent to the bulged nucleotide are selected to avoid alternative conformations. Both purines bind in a very similar way in a pocket in the protein. In the C-10 variant, which has very low affinity, the cytidine is partly inserted in the protein pocket rather than intercalated in the RNA stem. Substitution of the wild-type adenosine at position -7 by pyrimidines gives strongly reduced affinities, but the structure of the C-7 complex shows that the base occupies the same position as the A-7 in the wild-type RNA. It is stacked in the RNA and makes no direct contact with the protein.
authors.affiliation: Department of Cell and Molecular Biology, Uppsala University, Box 596, SE-751 24 Uppsala, Sweden.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Helgstrand
authors.forename: Elin
authors.initials: E
authors.lastname: Grahn
authors.forename: Timothy
authors.initials: T
authors.lastname: Moss
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Stonehouse
authors.forename: Kaspars
authors.initials: K
authors.lastname: Tars
authors.forename: Peter G
authors.initials: PG
authors.lastname: Stockley
authors.forename: Lars
authors.initials: L
authors.lastname: Liljas
mesh_terms: D015075:2-Aminopurine / Q000737:chemistry; D000241:Adenosine / Q000737:chemistry / Q000235:genetics; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D002213:Capsid / Q000737:chemistry / Q000378:metabolism*; D036022:Capsid Proteins; D003562:Cytidine / Q000737:chemistry / Q000235:genetics; D006151:Guanosine / Q000737:chemistry / Q000235:genetics; D006860:Hydrogen Bonding; D007288:Inosine / Q000737:chemistry; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D014176:Protein Biosynthesis; D011687:Purines / Q000737:chemistry*; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics
chemical_list: D036022:Capsid Proteins; D011687:Purines; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D006151:Guanosine; D015075:2-Aminopurine; D007288:Inosine; D003562:Cytidine; D012324:RNA-Dependent RNA Polymerase; D000241:Adenosine