ИСТИНА

eIF2D and its homologs MCTS1 and DENR are eukaryotic translation factors that have an ability to either facilitate tRNA binding to the P-site of the ribosome at the start codon, or release it from the post-termination complex at the stop codon. Recently, the factors were shown to affect uORF containing mRNA translation. However, their function in living cells remains uncertain. To dissect function of these factors in yeast, we prepared cell-free translation systems from three S.cerevisiae strains: wt; a double knockout strain lacking both TMA64 (EIF2D ortholog) and TMA20 (MCTS1 ortholog) genes; a double knockout strain lacking both TMA64 and TMA22 (DENR ortholog) genes. A set of luciferase encoding mRNAs with 5’ untranslated regions bearing different uORFs was prepared and translated in these in vitro systems. We observed an enhanced translation of the uORF containing luciferase mRNAs in the absence of the factors, in contrast to uORF-less ones. The effect depends on uORF length and a distance between uORF and the reporter coding sequence. An inadequately high translation level of the second cistron was detected also in the case of bicistronic Rluc-Fluc mRNAs. We concluded that reinitiation rate is highly increased in the absence of the factors. The rescue of Δtma64/Δtma20 and Δtma64/Δtma22 lysates by addition of the recombinant TMA20/TMA22 dimer or MCTS1/DENR dimer confirmed the conclusion. We proposed a model that these proteins operate regularly to prevent the post-termination complex at the 3’ end of the coding region from binding eIF1, eIF2-Met-tRNAi and eIF3 and thus to protect the recycled ribosomes from being recruited to a novel (unauthorized) round of translation initiation on the 3’ untranslated region. In some cases, however, they could allow this binding normally, thus discriminating between appropriate and illegitimate translation reinitiation on eukaryotic mRNAs. This work was supported by the Russian Science Foundation (RSF 14-50-00060).