Direct analysis of aminoacylation levels of tRNAs in vivo. Application to studying recognition of Escherichia coli initiator tRNA mutants by glutaminyl-tRNA synthetase.

U Varshney,C.P Lee,U.L Rajbhandary

doi:10.1016/s0021-9258(18)54288-5

U Varshney, C.P Lee + Show 1 more

Open Access

https://doi.org/10.1016/s0021-9258(18)54288-5

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Abstract

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.

Highlights

We describe the use of a gel electrophoretic method acidsinserted at therelevantsite.Anotherapproach for for measuring the levels of aminoacylation in vivo of studying the effect of mutations in a tRNA i n vivo is based mutant Escherichiacoli initiator tRNAs, which are on the efficiency in suppression of termination codons in a substrates for E. coli glutaminyl-tRNA synthetase mRNA
Except for one mutant, the observed levels of adaptation of a method, previously reported by Ho and Kan aminoacylationof the mutant tRNAs in vivoagree with [25], for measuring the levels of aminoacylation of several those expected on the basis of the kinetic parameters mutant E. coli initiator tRNAs with glutamine
Mutant tRNA genes were expressed in E. coli B, whichlackstRNApt 3 ; and tRNAs were purified by polyacrylamidegelelectrophoresis

Summary

A A c c GA CGAG

Hybridization responses for both the mutant initiator tRNAs and tyrosine tRNAs were essentially linear. The tRNAs were aminoacylated in vitro with glutamine using purified E. coli GlnRS to obtain the charged tRNAs[9]. Aliquots from these reactions were incubated with N"-formyltetrahydrofolate and an S-100 extract of E. coli [26, 33] to obtain the formylated tRNAs. Aliquots from these reactions were incubated with N"-formyltetrahydrofolate and an S-100 extract of E. coli [26, 33] to obtain the formylated tRNAs Both setsof reaction mixtures were extracted with phenol equilibrated with NaAc (pH 5.0), and nucleic acids were recovered by precipitation with ethanol. Conditions of work-up and gel electrophoresis do not result in deacylation of charged tRNAs. A mixture of all three forms of the T35A36 mutant initiator tRNA was routinely used as marker

RESULTS

41.2 T35A36

DISCUSSION