title: Water in the active site of an all-RNA hairpin ribozyme and effects of Gua8 base variants on the geometry of phosphoryl transfer.
abstract: The hairpin ribozyme requires functional group contributions from G8 to assist in phosphodiester bond cleavage. Previously, replacement of G8 by a series of nucleobase variants showed little effect on interdomain docking, but a 3-250-fold effect on catalysis. To identify G8 features that contribute to catalysis within the hairpin ribozyme active site, structures for five base variants were determined by X-ray crystallography in a resolution range between 2.3 and 2.7 A. For comparison, a native all-RNA "G8" hairpin ribozyme structure was refined to 2.05 A resolution. The native structure revealed a scissile bond angle (tau) of 158 degrees, which is close to the requisite 180 degrees "in-line" geometry. Mutations G8(inosine), G8(diaminopurine), G8(aminopurine), G8(adenosine), and G8(uridine) folded properly, but exhibited nonideal scissile bond geometries (tau ranging from 118 degrees to 93 degrees) that paralleled their diminished solution activities. A superposition ensemble of all structures, including a previously described hairpin ribozyme-vanadate complex, indicated the scissile bond can adopt a variety of conformations resulting from perturbation of the chemical environment and provided a rationale for how the exocyclic amine of nucleobase 8 promotes productive, in-line geometry. Changes at position 8 also caused variations in the A-1 sugar pucker. In this regard, variants A8 and U8 appeared to represent nonproductive ground states in which their 2'-OH groups mimicked the pro-R, nonbridging oxygen of the vanadate transition-state complex. Finally, the results indicated that ordered water molecules bind near the 2'-hydroxyl of A-1, lending support to the hypothesis that solvent may play an important role in the reaction.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Salter
authors.forename: Jolanta
authors.initials: J
authors.lastname: Krucinska
authors.forename: Shabnam
authors.initials: S
authors.lastname: Alam
authors.forename: Valerie
authors.initials: V
authors.lastname: Grum-Tokars
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Wedekind
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D007700:Kinetics; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D010758:Phosphorus / Q000737:chemistry* / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014867:Water / Q000378:metabolism*
chemical_list: D016337:RNA, Catalytic; C418481:hairpin ribozyme; D014867:Water; D010758:Phosphorus