Discrimination by Escherichia coli initiation factor IF3 against initiation on non-canonical codons relies on complementarity rules.

Abstract Translation initiation factor IF3, one of three factors specifically required for translation initiation in Escherichia coli , inhibits initiation on any codon other than the three canonical initiation codons, AUG, GUG, or UUG. This discrimination against initiation on non-canonical codons could be due to either direct recognition of the two last bases of the codon and their cognate bases on the anticodon or to some ability to “feel” codon-anticodon complementarity. To investigate the importance of codon-anticodon complementarity in the discriminatory role of IF3, we constructed a derivative of tRNA Leu that has all the known characteristics of an initiator tRNA except the CAU anticodon. This tRNA is efficiently formylated by methionyl-tRNA fMet transformylase and charged by leucyl-tRNA synthetase irrespective of the sequence of its anticodon. These initiator tRNA Leu derivatives (called tRNA LI ) allow initiation at all the non-canonical codons tested, provided that the complementarity between the codon and the anticodon of the initiator tRNA Leu is respected. More remarkably, the discrimination by IF3, normally observed with non-canonical codons, is neutralised if a tRNA LI carrying a complementary anticodon is used for initiation. This suggests that IF3 somehow recognises codon-anticodon complementarity, at least at the second and third position of the codon, rather than some specific bases in either the codon or the anticodon.