Shan, Shu-ou and Yoshida, Aiichiro and Sun, Sengen and Piccirilli, Joseph A. and Herschlag, Daniel (1999) Three metal ions at the active site of the Tetrahymena group I ribozyme. Proceedings of the National Academy of Sciences of the United States of America, 96 (22). pp. 12299-12304. ISSN 0027-8424. PMCID PMC22911. doi:10.1073/pnas.96.22.12299. https://resolver.caltech.edu/CaltechAUTHORS:20191004-154804302
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Abstract
Metal ions are critical for catalysis by many RNA and protein enzymes. To understand how these enzymes use metal ions for catalysis, it is crucial to determine how many metal ions are positioned at the active site. We report here an approach, combining atomic mutagenesis with quantitative determination of metal ion affinities, that allows individual metal ions to be distinguished. Using this approach, we show that at the active site of the Tetrahymena group I ribozyme the previously identified metal ion interactions with three substrate atoms, the 3′-oxygen of the oligonucleotide substrate and the 3′- and 2′-moieties of the guanosine nucleophile, are mediated by three distinct metal ions. This approach provides a general tool for distinguishing active site metal ions and allows the properties and roles of individual metal ions to be probed, even within the sea of metal ions bound to RNA.
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| Additional Information: | © 1999 The National Academy of Sciences. Communicated by Olke C. Uhlenbeck, University of Colorado, Boulder, CO, August 27, 1999 (received for review May 13, 1999). We are grateful to F. Eckstein for the gift of 2′-aminoguanosine, L. Beigelman for oligonucleotides, G. Narlikar for initial results and intellectual input, J. Brauman and members of the Herschlag laboratory for comments on the manuscript, and a reviewer for suggestions on the description of the experimental approach. This work was supported by grants from the National Institutes of Health to D. H. and the Howard Hughes Medical Institute to J.A.P.; S. Sun is a Research Associate of the Howard Hughes Medical Institute. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact. | |||||||||
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| Issue or Number: | 22 | |||||||||
| PubMed Central ID: | PMC22911 | |||||||||
| DOI: | 10.1073/pnas.96.22.12299 | |||||||||
| Record Number: | CaltechAUTHORS:20191004-154804302 | |||||||||
| Persistent URL: | https://resolver.caltech.edu/CaltechAUTHORS:20191004-154804302 | |||||||||
| Official Citation: | Three metal ions at the active site of the Tetrahymena group I ribozyme. Shu-ou Shan, Aiichiro Yoshida, Sengen Sun, Joseph A. Piccirilli, Daniel Herschlag. Proceedings of the National Academy of Sciences Oct 1999, 96 (22) 12299-12304; DOI: 10.1073/pnas.96.22.12299 | |||||||||
| Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | |||||||||
| ID Code: | 99104 | |||||||||
| Collection: | CaltechAUTHORS | |||||||||
| Deposited By: | Tony Diaz | |||||||||
| Deposited On: | 04 Oct 2019 22:54 | |||||||||
| Last Modified: | 16 Nov 2021 17:43 |
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