Single-Molecule Studies of Cognate and Near-Cognate Elongation in anin vitroEukaryotic Translation System

The ribosome plays a central role in translation of the genetic code into amino acid sequences during synthesis of polypeptides. During each cycle of peptide elongation, the ribosome must discriminate between correct and incorrect aminoacyl-tRNAs according to the codon present in its A-site. Ribosomes rely on a complex sequence of proofreading mechanisms to minimize erroneous selection of incorrect aminoacyl-tRNAs that would lead to mistakes in translation. These mechanisms have been studied extensively in prokaryotic organisms, but eukaryotic elongation is less well understood. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) with an