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Switching tRNA(Gln) identity from glutamine to tryptophan
M J Rogers1, T Adachi, H Inokuchi
1Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
Summary
The middle base of the anticodon in glutaminyl-tRNA (tRNA(Gln)) is crucial for accurate aminoacylation. Researchers created an opal suppressor tRNA(Gln) that inserts tryptophan, demonstrating altered amino acid identity and synthetase recognition.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The anticodon's middle base (U35) in tRNA(Gln) is key for accurate aminoacylation by glutaminyl-tRNA synthetase (GlnRS).
- Understanding tRNA identity and synthetase recognition is vital for genetic code accuracy.
Purpose of the Study:
- To investigate the role of anticodon base substitutions in tRNA(Gln) and their impact on aminoacylation specificity.
- To characterize an opal suppressor tRNA(Gln) and its amino acid insertion profile.
- To explore the evolutionary conservation of codon recognition.
Main Methods:
- Genetic selection and mutagenesis to isolate suppressor tRNAs.
- In vivo suppression assays and N-terminal sequencing of proteins.
- In vitro aminoacylation assays with mutated tRNA transcripts.
Main Results:
- An opal suppressor tRNA(Gln) with C35 (anticodon UCA) efficiently suppressed UGA codons, inserting predominantly tryptophan.
- Mutations in the anticodon or discriminator nucleotide affected suppressor efficiency and amino acid insertion.
- Escherichia coli Tryptophanyl-tRNA synthetase (TrpRS) recognized all three anticodon nucleotides, including C35.
Conclusions:
- Base substitutions in the tRNA anticodon can alter amino acid identity, demonstrating interchangeability between glutamine and tryptophan.
- Synthetase discrimination involves specific recognition of anticodon bases, particularly at position 35 (U35 for GlnRS, C35 for TrpRS).
- The conserved use of UGA for tryptophan suggests evolutionary maintenance of TrpRS recognition of the UCA anticodon.