During prokaryotic translation initiation, the small, 30S and large, 50S ribosomal subunits, along with formylmethionyl-transfer RNA (fMet-tRNAfMet), assemble at an authentic AUG messenger RNA (mRNA) start codon. The fidelity of initiation is regulated by three initiation factors (IFs) which kinetically control individual steps along the reaction pathway. One such step is recruitment of the 50S subunit to a 30S initiation complex (30SIC), composed of the 30S subunit, mRNA, tRNA, and IFs 1, 2, and 3. Although the GTPase IF2 has been shown to be principally responsible for selectively accelerating the rate of 50S subunit joining to a correctly initiated 30SIC, the molecular mechanism underlying this catalytic activity remains unknown. In order to elucidate the mechanism through which IF2 selectively catalyzes 50S subunit joining, we have developed single-molecule Forster resonance energy transfer (smFRET) signals between fluorescently-labeled IF2 and two tRNAs, an initiator fMet-tRNAfMet and an elongator Phe-tRNAPhe. Using these IF2-tRNA smFRET signals, we have characterized the dynamics of IF2-tRNA interactions within correctly and incorrectly initiated 30SICs. Our data reveal that the residence lifetime of IF2 on the 30SIC (τ30SIC), a parameter that directly controls IF2's ability to recruit the 50S subunit to the 30SIC, is highly regulated during initiation. We find that τ30SIC is exquisitely sensitive to: (1) the presence of IF1 and IF3, (2) the GTP- vs. GDP- or nucleotide-free forms of IF2, (3) the presence of fMet-tRNAfMet vs. Phe-tRNAPhe, and (4) the presence of a correct AUG start codon. Thus, only in the presence of IF1, IF3, GTP, and a correctly formed codon-anticodon interaction between an authentic AUG start codon and an initiator fMet-tRNAfMet is IF2 binding to the 30SIC significantly stabilized such that 50S subunit joining is efficiently catalyzed, ensuring the fidelity of this step of translation initiation.
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