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Mutations which alter the elbow region of tRNA 2 Gly reduce T4 gene 60 translational bypassing efficiency
Author(s) -
Herr Alan J.,
Atkins John F.,
Gesteland Raymond F.
Publication year - 1999
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/18.10.2886
Subject(s) - biology , gene , elbow , genetics , mutation , anatomy
Translating ribosomes bypass a 50 nucleotide coding gap in bacteriophage T4 gene 60 mRNA between codons 46 and 47 in order to synthesize the full‐length protein. Bypassing of the coding gap requires peptidyl‐tRNA 2 Gly detachment from a GGA codon (codon 46) followed by re‐pairing at a matching GGA codon just before codon 47. Using negative selection, based on the sacB gene from Bacillus subtilis , Escherichia coli mutants were isolated which reduce bypassing efficiency. All of the mutations are in the gene for tRNA 2 Gly . Most of the mutations disrupt the hydrogen bonding interactions between the D‐ and T‐loops (G18●ψ55 and G19●C56) which stabilize the elbow region in nearly all tRNAs. The lone mutation not in the elbow region destabilizes the anticodon stem at position 40. Previously described Salmonella typhimurium mutants of tRNA 2 Gly , which reduce the stability of the T‐loop, were also tested and found to decrease bypassing efficiency. Each tRNA 2 Gly mutant is functional in translation (tRNA 2 Gly is essential), but has a decoding efficiency 10‐ to 20‐fold lower than wild‐type. This suggests that rigidity of the elbow region and the anticodon stem is critical for both codon–anticodon stability and bypassing.