Modulation of tRNAAla identity by inorganic pyrophosphatase

Alexey D. Wolfson, Olke C. Uhlenbeck*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

102 Scopus citations

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 (kcat/KM) 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 kcat/KM 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.

Original languageEnglish (US)
Pages (from-to)5965-5970
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume99
Issue number9
DOIs
StatePublished - Apr 30 2002

ASJC Scopus subject areas

  • General

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