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

Translation initiation factor IF3, one of three factors specifically requir ed for translation initiation in Escherichia coli, inhibits initiation on a ny codon other than the three canonical initiation codons, AUG, GUG, or UUG . This discrimination against initiation on non-canonical codons could be d ue to either direct recognition of the two last bases of the codon and thei r cognate bases on the anticodon or to some ability to "feel" codon-anticod on complementarity. To investigate the importance of codon-anticodon comple mentarity in the discriminatory role of IF3, we constructed a derivative of tRNA(Leu) that has all the known characteristics of an initiator tRNA exce pt 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 (call ed tRNA(LI)) allow initiation at all the non-canonical codons tested, provi ded that the complementarity between the codon and the anticodon of the ini tiator tRNA(Leu) is respected. More remarkably, the discrimination by IF3, normally observed with non-canonical codons, is neutralised if a tRNA(LI) c arrying a complementary anticodon is used for initiation. This suggests tha t IF3 somehow recognises codon-anticodon complementarity, at least at the s econd and third position of the codon, rather than some specific bases in e ither the codon or the anticodon. (C) 1999 Academic Press.