Switching tRNA(Gln) identity from glutamine to tryptophan.
The middle base (U35) of the anticodon of tRNA(Gln) is a major element ensuring the accuracy of aminoacylation by Escherichia coli glutaminyl-tRNA synthetase (GlnRS). An opal suppressor of tRNA(Gln) (su+2UGA) containing C35 (anticodon UCA) was isolated by genetic selection and mutagenesis. Suppression of a UGA mutation in the E. coli fol gene followed by N-terminal sequence analysis of purified dihydrofolate reductase showed that this tRNA was an efficient suppressor that inserted predominantly tryptophan. Mutations of the 3-70 base pair (U70 and A3U70) were made. These mutants of su+2UGA are less efficient suppressors and inserted predominantly tryptophan in vivo; alanine insertion was not observed. Mutations of the discriminator nucleotide (A73, U73, C73) result in very weak opal suppressors. Aminoacylation in vitro by E. coli TrpRS of tRNA(Gln) transcripts mutated in the anticodon demonstrate that TrpRS recognizes all three nucleotides of the anticodon. The results show the interchangeability of the glutamine and tryptophan identities by base substitutions in their respective tRNAs. The amber suppressor (anticodon CUA) tRNA(Trp) was known previously to insert predominantly glutamine. We show that the opal suppressor (anticodon UCA) tRNA(Gln) inserts mainly tryptophan. Discrimination by these synthetases for tRNA includes position 35, with recognition of C35 by TrpRS and U35 by GlnRS. As the use of the UGA codon as tryptophan in mycoplasma and in yeast mitochondria is conserved, recognition of the UCA anticodon by TrpRS may also be maintained in evolution.
Rogers
M J
MJ
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
Adachi
T
T
Inokuchi
H
H
Söll
D
D
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D000926:Anticodon / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D016153:Genes, Suppressor; D005813:Genes, Synthetic; D005973:Glutamine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D012351:RNA, Transfer, Gln / Q000235:genetics / Q000378:metabolism*; D013489:Suppression, Genetic; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014364:Tryptophan / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000926:Anticodon; D012351:RNA, Transfer, Gln; D005973:Glutamine; D014364:Tryptophan; D013762:Tetrahydrofolate Dehydrogenase; D001616:beta-Galactosidase; D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase