| Summary: | 碩士 === 國立中央大學 === 生命科學研究所 === 92 === There are two kinds of non-specific tRNA-binding proteins in the eukaryotic cell. The first kind of non-specific tRNA-binding protein participates in physiological functions of the cell; an example is Arc1p, which improves the binding affinity between aminoacyl-tRNA synthetases and their cognate tRNAs. The other kind of non-specific tRNA-binding protein plays a part in tRNA export; for example, Utp8p is involved in nuclear tRNA export. However, current knowledge of tRNA-binding proteins is still very limited. Therefore, we chose the three-hybrid screening system (using tRNAGln as bait) to gain insight into other tRNA binding proteins in vivo. After screening a yeast genomic DNA library, the positive candidates were classified into nineteen groups based on the size of the library fragment. Six groups, representing the groups with the most clones or groups with the strongest interaction between bait and prey, were chosen for sequencing. After BLAST searching the sequences against the database, two kinds of results were achieved. In four of the clones, an in-frame open reading frame (ORF) corresponding to the sequence of the library fragment could not be found in the yeast database; in other words, these peptides did not exist. The other clones were successfully matched to in-frame ORFs in the yeast database― one was GCR2, a transcriptional activator of glycolytic genes, and the other was Yol155c, a cell wall protein. After appropriate deletion of unnecessary fragments, we retested the clones by the three-hybrid system and found the library-bait interaction to still be positive. We will purify these library peptides and further test for tRNA binding ability in vitro. These results will give us a more in-depth understanding of what roles they play in vivo.
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