Mutations which alter the elbow region of tRNA(2)(Gly) reduce T4 gene 60 translational bypassing efficiency

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-leng th protein. Bypassing of the coding gap requires peptidyl-tRNA(2)(Gly) deta chment 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 ge ne from Bacillus subtilis, Escherichia coli mutants were isolated which red uce bypassing efficiency. All of the mutations are in the gene for tRNA(2)( Gly) Most of the mutations disrupt the hydrogen bonding interactions betwee n the D- and T-loops (G18.psi 55 and G19.C56) which stabilize the elbow reg ion in nearly all tRNAs, The lone mutation not in the elbow region destabil izes the anticodon stem at position 40, Previously described Salmonella typ himurium 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)(G ly) mutant is functional in translation (tRNA(2)(Gly) is essential), but ha s 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 bo th codon-anticodon stability and bypassing.