Abstract:
The kinetic mechanism of mRNA decoding has been established through ensemble kinetic investigations using purified translation components from Escherichia coli. Pre-steady state single-molecule fluorescence resonance energy transfer (smFRET) measurements have directly revealed insights into the movement of aminoacyl-tRNA (aa-tRNA) within the A site of the ribosome and their mRNA codon-dependencies. Here, we have interrogated the conformational dynamics of aa-tRNA on the ribosome during mRNA decoding using a multiplexed smFRET approach that facilitates side-by-side comparisons of multiple samples under identical experimental conditions. We demonstrate the utility of this approach by simultaneously examining the process of elongation factor-Tu (EF-Tu) facilitated Phe-tRNAPhe incorporation into 4 distinct initiation complexes bearing either cognate or near-cognate codons in the A site. As expected from prior ensemble investigations, our results show that the cognate Phe codons, UUU and UUC, exhibit distinctions in the rates of tRNA selection and those near-cognate codons (CUC and UCC) efficiently reject Phe-tRNAPhe during early steps of tRNA selection prior to GTP hydrolysis. We then examined the impacts of the miscoding antibiotic streptomycin on mRNA decoding. Prior ensemble investigations report that streptomycin slows cognate tRNA selection and accelerates near-cognate tRNA selection during intermediate steps of mRNA decoding to homogenize the rates of GTP hydrolysis and peptide bond formation. Our findings show that the primary impact of streptomycin is to accelerate the rates of mRNA decoding for both cognate and near-cognate ribosome complexes, while slowing the rates of aa-tRNA binding to the A site of the ribosome. These data are consistent with streptomycin favoring “closed” conformational states of the small subunit shoulder domain prior to aa-tRNA binding that inhibit early codon recognition steps where the tRNA anticodon is escorted by EF-Tu into the A site decoding region on the small ribosomal subunit.