Discrimination of 5′-terminal start codons by translation initiation factor 3 is mediated by ribosomal protein S1

Abstract

The interrelation between ribosomal protein S1 and IF3 in recognition/discrimination of 5′-terminal start codons by 30S ribosomes has been studied using in vitro toeprinting. The study has been performed with two naturally occurring leaderless mRNAs, λ cI and phage r1t rro mRNA, as well as with an artificial leaderless mRNA derived from the E. coli ompA gene. We show that in the absence of S1, IF3 does not discriminate against the authentic 5′-terminal start codon of both cI and rro mRNA. Since IF3 was able to exert its proofreading function for initiator tRNA Met f on 30S ribosomes lacking S1, this observation cannot be attributed to a lack of binding to or action of IF3 on 30S(−S1) ribosomes. In contrast to leaderless mRNAs, ternary complex formation occurs in the presence of IF3 with 30S ribosomes when the start codon is preceded by a short 20-nucleotide 5′-untranslated region containing a canonical Shine and Dalgarno sequence. This suggests that 5′-terminal start codons are recognised by IF3 as non-standard because of the lack of 16S rRNA-mRNA contacts.