| Summary: | 碩士 === 國立中央大學 === 生命科學研究所 === 91 === ABSTRACT
GRS1 was previously identified as the only gene coding for glycyl-tRNA synthetase activity in the yeast Saccharomyces cerevisiae. Evidence presented here shows that two distinct protein isoforms are generated from this gene: a short cytoplasmic form, which is translationally initiated at the putative ATG initiator (i.e., ATG1), and a longer mitochondrial form, which is initiated at an upstream in-frame non-ATG codon (i.e., TTG-23). A reverse transcription approach in conjunction with Western blot analysis suggests that the isoforms are translated via leaky scanning from a single transcript of this gene, which has its 5’-end located at position —88 relative to ATG1. Although the isoforms have essentially the same polypeptide sequence, they cannot substitute for each other because of different localization. A domain fusion study suggests that the leader peptide of the mitochondrial isoform can direct a cytoplasmic passenger into mitochondria, while the cytoplasmic form of glycyl-tRNA synthetase can be converted into a mitochondrial protein by fusion of a heterologous signal peptide, suggesting that it is the nature of the leader peptide that is responsible for the subcellular localization of the isoforms.
A second part of the thesis is focused on the mechanism of non-ATG initiation in yeast. Using a reporter gene assay, we show that redundant non-ATG codons have a higher efficiency of translation initiation than a single non-ATG codon at both qualitative and quantitative levels. Even more remarkably, redundant non-ATG codons can substitute for the initiating activity of the ATG initiator of the reporter gene. Thus, the results present a novel mechanism by which the initiating activity of a weak start site can be significantly improved. As in many cases of non-ATG initiation in higher eukaryotes, a stable RNA secondary structure that is predicted to enhance the initiating activity of the non-ATG initiator is found downstream of the GRS1 TTG-23. Introduction of a short sequence between TTG-23 and the secondary structure impairs its initiating activity, suggesting that the distance between the non-ATG initiator and secondary structure is critical for recognition of the weak start site. Our mutagenesis study further shows that, as in higher eukaryotes, many non-ATG codons that differ from ATG by just one nucleotide can be used as translation initiators in yeast. Most surprisingly, an AGC codon, which differs from ATG by two nucleotides, is also functional as an initiator under the conditions used. To our knowledge, this appears to be the first example where a non-ATG triplet that differs from ATG by two nucleotides can still serve as a translation start site.
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