大腸桿菌alanyl-tRNA synthetase（AlaRS）的胺基酸序列可以約略分成2個功能區域：N端是酵素活化區可以進行胺醯化作用（aminoacylation）；C端則與此酵素形成多聚體（oligomerization）有關。將目前已知不同物種的AlaRS進行胺基酸初級結構比對，發現其N端具有高度的保留性，因此可能是演化早期即已存在；而C端其保留性非常的低，可能是演化後期才被加入的部分。在生化活性方面，N端AlaRS與全長AlaRS對microhelix（tRNAAla acceptor stem的部分）的催化能力相近。加上先前的研究結果，一般認為AlaRS對tRNAAla專一性的辨識位置是位在N端（胺基酸1-461），C端則是增加此酵素與tRNA的非專一性結合能力。 因此我們想要利用AlaRS的N端及一些非專一性的tRNA結合蛋白，來組合一個在活體中具有AlaRS功能的蛋白質。我們的結果顯示，大腸桿菌的AlaRS 位於C端形成多倍體區域在細胞中是必需的。不論是大腸桿菌AlaRS的N端胺基酸1-461或是大腸桿菌AlaRS的N端胺基酸1-699，皆不能補償大腸桿菌AlaRS基因（alaS）刪除株。而融合蛋白質（非專一性tRNA結合蛋白質與大腸桿菌AlaRS的N端胺基酸1-461或是大腸桿菌AlaRS的N端胺基酸1-699的組合）也不能明顯的互補大腸桿菌AlaRS基因（alaS）刪除株。酵母菌的融合蛋白質也不能補償酵母菌AlaRS基因（ALA1）刪除株。但是有趣的是我們發現，將酵母菌細胞質的AlaRS基因構築在具ADH promoter的載體上時，不但可以補償酵母菌AlaRS細胞質刪除株的功能，同時也可以補償酵母菌粒線體刪除株的功能。因此我們推測，在酵母菌中，一個ALA1基因會轉譯出具有細胞質及粒線體雙重功能的AlaRS。 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.
