Record number :
1158225
Title of article :
The role of the cleavage site 2′-hydroxyl in the Tetrahymena group I ribozyme reaction Original Research Article
Author/Authors :
Aiichiro Yoshida، نويسنده , , Shu-ou Shan، نويسنده , , Daniel Herschlag، نويسنده , , Joseph A. Piccirilli، نويسنده ,
Issue Information :
ماهنامه با شماره پیاپی سال 2000
Pages :
12
From page :
85
To page :
96
Abstract :
Abstract Background: The 2′-hydroxyl of U preceding the cleavage site, U(−1), in the Tetrahymena ribozyme reaction contributes 103-fold to catalysis relative to a 2′-hydrogen atom. Previously proposed models for the catalytic role of this 2′-OH involve coordination of a catalytic metal ion and hydrogen-bond donation to the 3′-bridging oxygen. An additional model, hydrogen-bond donation by the 2′-OH to a nonbridging reactive phosphoryl oxygen, is also consistent with previous results. We have tested these models using atomic-level substrate modifications and kinetic and thermodynamic analyses. Results: Replacing the 2′-OH with -NH3+ increases the reaction rate ∼60-fold, despite the absence of lone-pair electrons on the 2′-NH3+ group to coordinate a metal ion. Binding and reaction of a modified oligonucleotide substrate with 2′-NH2 at U(−1) are unaffected by soft-metal ions. These results suggest that the 2′-OH of U(−1) does not interact with a metal ion. The contribution of the 2′-moiety of U(−1) is unperturbed by thio substitution at either of the nonbridging oxygens of the reactive phosphoryl group, providing no indication of a hydrogen bond between the 2′-OH and the nonbridging phosphoryl oxygens. In contrast, the 103-fold catalytic advantage of 2′-OH relative to 2′-H is eliminated when the 3′-bridging oxygen is replaced by sulfur. As sulfur is a weaker hydrogen-bond acceptor than oxygen, this effect suggests a hydrogen-bonding interaction between the 2′-OH and the 3′-bridging oxygen. Conclusions: These results provide the first experimental support for the model in which the 2′-OH of U(−1) donates a hydrogen bond to the neighboring 3′-bridging oxygen, thereby stabilizing the developing negative charge on the 3′-oxygen in the transition state.
Keywords :
* Mechanistic analysis , * RNA catalysis , * Chemical modification
Journal title :
Chemistry and Biology
Serial Year :
2000
Link To Document :
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