Modulation of tRNAAla identity by inorganic pyrophosphatase.
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'-(32)P-labeled tRNA. When applied to Escherichia coli alanyl-tRNA synthetase, the assay allowed accurate measurement of aminoacylation of the most deleterious mutants of tRNA(Ala). The effect of tRNA(Ala) 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 tRNA(Ala) identity.
Wolfson
Alexey D
AD
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA.
Uhlenbeck
Olke C
OC
D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D009154:Mutation; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D011755:Pyrophosphatases / Q000378:metabolism*; D012344:RNA, Transfer, Ala / Q000378:metabolism*; D013997:Time Factors
D012344:RNA, Transfer, Ala; D010444:Peptide Elongation Factor Tu; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase