Futernyk P. Non-canonical interactions of translation apparatus components of higher eukaryotes

Thesis is devoted to the study on possibility of stable complexes formation between non-charged tRNAs and eEF1A*GDP as well as to experimental substantiation of a hypothesis concerning universality of the molecular mechanisms of tRNA channeling in the course of protein synthesis, which lies in the formation of stable complexes of initiation and elongation factors with corresponding tRNA. The experimental data obtained confirm the formation of stable complexes of 10 non-charged tRNAs with eEF1A*GDP. The affinity of these complexes was analyzed using the band shift assay. The tissue specific isoform eEF1A2 from rabbit muscles was found to form 2-4 times more stable complexes with tRNA than eEF1A1 from rabbit liver. The formation of the yeast eEF1A*GDP complex with non-charged tRNAs was revealed by two methods including FRET and band shift assay. It was demonstrated the principal possibility of high affinity association between translation initiation factor eIF2*GDP with deacylated tRNAiMet. The eEF1A*GDP from higher eukaryotes was found to interact with initiator deacylated tRNAіМеt forming the complex with stability equal to that with elongator tRNAs. Using site-directed mutagenesis we established that the changes in Т-stem of recombinant tRNA3Lys in sites, which are supposed to participate in the ternary complex formation, only slightly decreased the affinity to the factor, preferentially to the isoform eEF1A2. The formation of non-canonical quaternary complex [SerRS*tRNASer*eEF1A*GDP] analogous to found earlier the quaternary complex [PheRS*tRNAPhe*eEF1A*GDP] was shown. In addition, the stabilizing effect of eEF1A*GDP on complex [TyrRS- С*tRNATyr] was revealed. Thus, our data about the formation of stable complexes of mammalian and yeast eEF1A and eIF2 with uncharged tRNAs are in favor of universality of the molecular mechanisms of tRNA channeling in protein biosynthesis in eukaryotic cells Key words: eukaryotic elongation translation factor 1A, eukaryotic initiation factor 2, tRNA, aminoacyl-tRNA synthetase, channeling.