Modulation of tRNAAla identity by inorganic pyrophosphatase
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Contributed by Olke C. Uhlenbeck
Abstract
A highly sensitive assay of tRNA aminoacylation was developed that directly measures the fraction of aminoacylated tRNA by following amino acid attachment to the 3′-32P-labeled tRNA. When applied to Escherichia coli alanyl-tRNA synthetase, the assay allowed accurate measurement of aminoacylation of the most deleterious mutants of tRNAAla. The effect of tRNAAla identity mutations on both aminoacylation efficiency (k cat/K M) and steady-state level of aminoacyl-tRNA was evaluated in the absence and presence of inorganic pyrophosphatase and elongation factor Tu. Significant levels of aminoacylation were achieved for tRNA mutants even when the k cat/K M value is reduced by as much as several thousandfold. These results partially reconcile the discrepancy between in vivo and in vitro analysis of tRNAAla identity.
Footnotes
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↵ * To whom reprint requests should be addressed at: Department of Chemistry and Biochemistry, 215 UCB, University of Boulder, Boulder, CO 80309-0215. E-mail: Olke.Uhlenbeck{at}Colorado.edu.
- Abbreviations:
- aa-tRNA,
- aminoacyl-tRNA;
- aaRS,
- aminoacyl-tRNA synthetase;
- AlaRS,
- alanyl-tRNA synthetase;
- PPase,
- inorganic pyrophosphatase;
- EF-Tu,
- translational elongation factor Tu;
- PheRS,
- phenylalanyl-tRNA synthetase
- Copyright © 2002, The National Academy of Sciences
