| Summary: | 碩士 === 國立中央大學 === 生命科學研究所 === 89 === The polypeptide sequence of E. coli alanyl-tRNA synthetase (AlaRS) can be divided into two functional domains: an N-terminal domain, which is necessary and sufficient for aminoacylation, and a C-terminal domain, which is involved in oligomerization of the enzyme. Primary sequence analyses show that the N-terminal domain is highly conserved among all known AlaRSs and is believed to be related through evolution, while the C-terminal domain shares relatively low homology among the alanine enzymes and is thought to be added to the molecule late in evolution. As a consequence, the N-terminal domain of AlaRS exhibits a catalytic activity similar to that of the full-length enzyme towards a microhelix substrate based on the acceptor stem sequence of tRNAAla. These results and others suggest that the specificity determinants of AlaRS for recognition of tRNAAla lie mainly in the N-terminal domain.
We are motivated to ask whether we could assemble an alanyl-tRNA synthetase that is active in vivo, using the N-terminal domain of AlaRS and nonspecific RNA binding domains. Our results show that the C-terminal oligomerization domain (residue 700-875) of E. coli AlaRS is essential for its in vivo function. Neither N461 (containing residue 1 to 461) nor N699 (containing residue 1 to 699) can complement an alaS (the gene coding for E. coli AlaRS) knockout strain. Fusion of a nonspecific RNA binding domain to either N461 or N699 has no significant effect on its complementing activity. Similar results were obtained using yeast AlaRS as a template for construction of fusion proteins. Interestingly, we found that the putative open reading frame for the yeast cytoplasmic AlaRS, when cloned in a high-copy-number vector under the control of a constitutive ADH promoter, could complement both the cytoplasmic and mitochondrial defects of an ALA1 (the gene coding for yeast AlaRS) disrupted allele, suggesting that a single ALA1 gene codes for both the cytoplasmic and mitochondrial functions of a AlaRS in yeast.
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