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

Abstract

Initiation factor-free 30S subunits of E. coli ribosomes bind aminoacyl-tRNAs more efficiently than fMet-tRNA(fMet). 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(fMet). 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(fMet) and with tRNA(fMet), fMet-tRNA(fMet) reacted 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(fMet) or tRNA(fMet) filled 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.