A proposed role for IF-3 and EF-T in maintaining the specificity of prokaryotic initiation complex formation

Initiation factor-free 30S subunits of E. coli ribosomes bind aminoacyl-tRNAs more efficiently than fMet-tRNA_inff^supMet. Elongator-tRNA binding was unaffected by IF-1 or IF-2 but was inhibited by IF-3. Their combination reduced this binding up to 40% and stimulated that of fMet-tRNA_inff^supMet. Unexpectedly, EF-T also prevented elongator-tRNA binding by complexing both to the 30S and to the aminoacyl-tRNAs. Using AUGU₃ as mRNA, elongator-tRNAs competed with fMet-fRNA_inff^supMetand with tRNA_inff^supMet. fMet-tRNA_inff^supMetreacted with puromycin after addition of 50S subunits suggesting that it occupied the P site. EF-T directed binding of phe-tRNA to the 30S.AUGU₃ complex at the A site only if fMet-tRNA_inff^supMetor tRNA_inff^supMetfilled the P/E site. We propose that one function of EF-T may be to prevent the entry of aminoacyl-tRNAs into the 30S particle during initiation. The possibility that a special site for fMet-tRNA resides on 16S rRNA is also discussed.