期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2002
卷号：99
期号：9
页码：5965-5970
DOI：10.1073/pnas.092152799
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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.
