Near-cognate tRNAs enhance tRNA rejection and prime the ribosome for rapid subsequent tRNA testing

The ribosome plays a central role in translating the genetic code into amino acid sequences during polypeptide synthesis. In each cycle of peptide elongation, the ribosome discriminates between correct and incorrect aminoacyl-tRNAs based on the codon present in its A-site. To ensure high fidelity, ribosomes employ multiple proofreading mechanisms that reduce the selection of incorrect aminoacyl-tRNAs. Initial proofreading of incorrect tRNAs (near-cognate or non-cognate) is well understood in prokaryotic ribosomes but incompletely understood in eukaryotic systems. To investigate tRNA selection and accommodation for both cognate and near-cognate tRNAs, we employed single-molecule fluorescence resonance energy transfer with an in vitro eukaryotic translation system assembled on an error-prone mRNA sequence. We compared tRNA binding and accommodation rates of tryptophan-aminoacyl-tRNA in the ribosomal A-site containing either cognate or near-cognate codons. Although initial sampling of near-cognate tRNAs was slower than for cognate tRNA, subsequent near-cognate sampling events proceeded more rapidly than the initial near-cognate event. Increasing the concentration of near-cognate aminoacyl-tRNA surprisingly decreased its accommodation efficiency, a phenomenon not seen with cognate tRNAs. These findings suggest that rejection of a near-cognate tRNA induces an altered ribosomal conformation that enhances discrimination against further errors while simultaneously accelerating the tRNA sampling rate.