Identity of Saccharomyces cerevisiae tRNA(Trp) is not changed by an anticodon mutation that creates an amber suppressor.

K.D Yesland,A.W Nelson,D.M Six Feathers,J.D Johnson

doi:10.1016/s0021-9258(18)54137-5

Abstract

A C35-->T mutation in an Escherichia coli tRNA(Trp) gene creates an amber suppressor which efficiently inserts glutamine in response to UAG codons in vivo (Soll, L., and Berg, P. (1969) Nature 223, 1340-1342). We have introduced the same change in a yeast tRNA(Trp) gene and demonstrated that the tRNA acts as an efficient amber suppressor in vivo. Amino acid sequence analyses were performed on chitinase produced by cells carrying the corresponding gene with a UAG codon at position 8 of the mature protein plus the mutant tRNA(Trp) gene. In contrast to comparable experiments with E. coli, tryptophan is inserted at a frequency > or = 80% by the yeast suppressor tRNA(Trp). Furthermore, in vitro charging experiments with the mutant tRNA(Trp) reveal no detectable increase in glutamine acceptor activity results from the C35-->T transition. The identity elements in E. coli tRNA(Gln) are well characterized (Jahn, M., Rogers, J., and Soll, D. (1991) Nature 352, 258-260). Sequence comparisons of the tRNA(Trp) and tRNA(Gln) molecules from E. coli reveal that the amber suppressor tRNA(Trp) has four of five identity elements required for glutaminyl-tRNA synthetase recognition. A similar comparison in the yeast system shows only two of the five potential identity elements are present. We conclude that, in spite of substantial structural similarities between yeast and E. coli aminoacyl-tRNA synthetases, fundamental differences can exist with regard to tRNA recognition.

Highlights

A C35+T mutation in an Escherichia coli tRNATm uitro (Yaniv et al, 1974; Himeno et al, 1991)
We have introduced the same change in a yeast tRNATrpgene and demonstrated that the tRNA acts as an efficient amber suppressorin vivo
In contrast to comparable experiments with E. coli, tryptophan is inserted at a frequency ~ 8 0 b%y the yeast suppressor tRNATrFpu. rthermore, in vitro charging experiments with the mutant tRNATmreveal no detectable increase in glutathat an ambersuppressor form of E. coli tRNATwinserts predominantlyGln duringtranslation of acorresponding nonsense codon position i n vivo

Summary

Introduction

A C35+T mutation in an Escherichia coli tRNATm uitro (Yaniv et al, 1974; Himeno et al, 1991). In support of this hypothesis, a G1 + A mutant in an amber suppressor form of E. coli tRNA5’ inserts Gln at UAG codons in vivo (Ghysen and Celis, 1974), suggesting that this base,along with U35 and G2:C71, are sufficient for positive recognition by GlnRS.

Results

Conclusion