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Investigation of adenosine base ionization in the hairpin ribozyme by nucleotide analog interference mapping

Published online by Cambridge University Press:  24 February 2017

SEAN P. RYDER
Affiliation:
Yale University, Department of Molecular Biophysics and Biochemistry, New Haven, Connecticut 06520-8114, USA
ADEGBOYEGA K. OYELERE
Affiliation:
Yale University, Department of Molecular Biophysics and Biochemistry, New Haven, Connecticut 06520-8114, USA
JENIFER L. PADILLA
Affiliation:
Yale University, Department of Molecular Biophysics and Biochemistry, New Haven, Connecticut 06520-8114, USA
DAGMAR KLOSTERMEIER
Affiliation:
The Scripps Research Institute, Department of Molecular Biology, La Jolla, California 92037, USA
DAVID P. MILLAR
Affiliation:
The Scripps Research Institute, Department of Molecular Biology, La Jolla, California 92037, USA
SCOTT A. STROBEL
Affiliation:
Yale University, Department of Molecular Biophysics and Biochemistry, New Haven, Connecticut 06520-8114, USA
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Abstract

Tertiary structure in globular RNA folds can create local environments that lead to pKa perturbation of specific nucleotide functional groups. To assess the prevalence of functionally relevant adenosine-specific pKa perturbation in RNA structure, we have altered the nucleotide analog interference mapping (NAIM) approach to include a series of α phosphorothioate-tagged adenosine analogs with shifted N1 pKa values. We have used these analogs to analyze the hairpin ribozyme, a small self-cleaving/ligating RNA catalyst that is proposed to employ a general acid-base reaction mechanism. A single adenosine (A10) within the ribozyme active site displayed an interference pattern consistent with a functionally significant base ionization. The exocyclic amino group of a second adenosine (A38) contributes substantially to hairpin catalysis, but ionization of the nucleotide does not appear to be important for activity. Within the hairpin ribozyme crystal structure, A10 and A38 line opposite edges of a solvent-excluded cavity adjacent to the 5′-OH nucleophile. The results are inconsistent with the model of ribozyme chemistry in which A38 acts as a general acid–base catalyst, and suggest that the hairpin ribozyme uses an alternative mechanism to achieve catalytic rate enhancement that utilizes functional groups within a solvent-excluded cleft in the ribozyme active site.

Type
Research Article
Information
RNA , Volume 7 , Issue 10 , October 2001 , pp. 1454 - 1463
Copyright
2001 RNA Society

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Investigation of adenosine base ionization in the hairpin ribozyme by nucleotide analog interference mapping
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