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

Ryder, S. P. and Oyelere, A. K. and Padilla, J. L. and Klostermeier, D. and Millar, D. P. and Strobel, S. A.. (2001) Investigation of adenosine base ionization in the hairpin ribozyme by nucleotide analog interference mapping. RNA, Vol. 7, H. 10. pp. 1454-1463.

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Official URL: http://edoc.unibas.ch/dok/A5259720

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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 a 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.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Biophysical Chemistry (Klostermeier)
UniBasel Contributors:Klostermeier, Dagmar
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:Cambridge University Press
Note:Publication type according to Uni Basel Research Database: Journal article
Last Modified:22 Mar 2012 14:21
Deposited On:22 Mar 2012 13:23

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