SUMMARY We propose to determine i) the specific effects on eukaryotic translation of posttranscriptional modifications (PTMs) of mRNA and tRNA and ii) how readthrough of premature termination codons (PTCs) is modulated by downstream sequence context and stimulated by translation readthrough inducing drugs (TRIDs). In so doing we will contribute to the development of new therapeutic approaches for treatment of diseases which are linked to PTMs and PTCs. Our approach is to apply a reconstituted in vitro system, denoted PURE‐LITE, to elucidate mechanisms of elongation and termination using a combination of ensemble and single molecule approaches. Such studies can aid in interpreting the results of related cellular and clinical studies and suggest new directions to pursue. Our Specific Aims are: 1. Determine the effects of mRNA and tRNA posttranscriptional modifications on elongation and termination. m6A, pseudoU (,inosine (I) and m5C are among the most common PTMs of eukaryotic mRNA. There currently are no direct measurements of how these PTMs affect cognate elongation, misreading, readthrough and termination. We will determine which modifications have the most pronounced effects on these processes and identify which steps during elongation and termination are responsible for these effects, which are the most likely to be involved in altering translational functions in cells. We will prepare mRNAs containing modifications in sense or nonsense codons, pair these modified codons with cognate and near‐ cognate tRNAs for the three elongation processes, and compare the rates and stoichiometries of elongation with those obtained with the corresponding unmodified mRNAs and tRNAs. tRNAs will be selected to examine how different modifications of nucleotides 34 and 37 of the anticodon loop affect assay results. Similar studies for termination will compare modified and unmodified mRNAs. 2. Determine the effects on readthrough of UGA codons of downstream sequence context and TRID termination inhibitors. There is strong evidence that UGA PTCs are the most susceptible to readthrough and that sequences immediately downstream from a UGA PTC influence readthrough efficiency. Our results have demonstrated that ataluren, the only TRID currently approved for treating patients with a PTC disease, acts exclusively by competitively inhibiting release factor complex (RFC). Ataluren shows variable results in stimulating readthrough, as measured in cell‐based assays and clinical trials. We hypothesize that this variability may be a consequence of how PTC downstream sequence affects RFC activity. We will test this hypothesis by determining how closely ataluren inhibition of RFC as a function of downstream sequence correlates with ataluren stimulation of readthrough activity. Such tests will be repeated with ataluren added in combination with agents which stimulate readthrough by a mechanism orthogonal to that of ataluren, potentially leading to synergistic results. The most...