TY - JOUR
T1 - Water in the active site of an All-RNA hairpin ribozyme and effects of Gua8 base variants on the geometry of phosphoryl transfer
AU - Salter, Jason
AU - Krucinska, Jolanta
AU - Alam, Shabnam
AU - Grum-Tokars, Valerie
AU - Wedekind, Joseph E.
N1 - Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2006/1/24
Y1 - 2006/1/24
N2 - 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 Å resolution. The native structure revealed a scissile bond angle (τ) of 158°, which is close to the requisite 180° "in-line" geometry. Mutations G8(inosine), G8(diaminopurine), G8(aminopurine), G8(adenosine), and G8(uridine) folded properly, but exhibited nonideal scissile bond geometries (τ ranging from 118° to 93°) 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.
AB - 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 Å resolution. The native structure revealed a scissile bond angle (τ) of 158°, which is close to the requisite 180° "in-line" geometry. Mutations G8(inosine), G8(diaminopurine), G8(aminopurine), G8(adenosine), and G8(uridine) folded properly, but exhibited nonideal scissile bond geometries (τ ranging from 118° to 93°) 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.
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U2 - 10.1021/bi051887k
DO - 10.1021/bi051887k
M3 - Article
C2 - 16411744
AN - SCOPUS:31044455894
VL - 45
SP - 686
EP - 700
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 3
ER -