Direct analysis of aminoacylation levels of tRNAs in vivo. Application to studying recognition of Escherichia coli initiator tRNA mutants by glutaminyl-tRNA synthetase.
We describe the use of a gel electrophoretic method for measuring the levels of aminoacylation in vivo of mutant Escherichia coli initiator tRNAs, which are substrates for E. coli glutaminyl-tRNA synthetase (GlnRS) due to an anticodon sequence change. Using this method, we have compared the effects of introducing further mutations in the acceptor stem, at base pairs 1:72, 2:71, and 3:70 and discriminator base 73, on the recognition of these tRNAs by E. coli GlnRS in vitro and in vivo. The effects of the acceptor stem mutations on the kinetic parameters for aminoacylation of the mutant tRNAs in vitro are consistent with interactions seen between this region of tRNA and GlnRS in the crystal structure of tRNA(Gln). GlnRS complex. Except for one mutant, the observed levels of aminoacylation of the mutant tRNAs in vivo agree with those expected on the basis of the kinetic parameters obtained in vitro. We have also measured the relative amounts of aminoacyl-tRNAs for the various mutants and their activities in suppression of an amber codon in vivo. We find that there is, in general, a good correlation between the relative amounts of aminoacyl-tRNAs and their activities in suppression.
Varshney
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Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Lee
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CP
RajBhandary
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UL
D000215:Acylation; D000588:Amines / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D003062:Codon; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D005975:Glutamate-tRNA Ligase / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational / Q000235:genetics*; D012343:RNA, Transfer / Q000378:metabolism*
D000588:Amines; D003062:Codon; D004269:DNA, Bacterial; D012343:RNA, Transfer; D005975:Glutamate-tRNA Ligase