Aminoacyl-tRNA synthetases (aaRSs)是一群必要的轉譯酵素，它們的主要功能是將胺基酸接到相對應的tRNA (胺醯化反應)，形成aminoacyl-tRNA，接著aminoacyl-tRNA會被延長因子攜帶至核醣體進行蛋白質合成。aaRSs主要是透過tRNA上的識別決定基(identity elements)來辨認相對應的tRNA。識別決定基多數集中在acceptor stem及anticodon loop上，不同的tRNA，含有不同的識別決定基。一般而言，tRNA的識別決定基在演化上高度保留，不會因物種來源不同而有所差異，例如Alanine tRNA (tRNAAla)的識別決定基是在acceptor stem上的一個G3:U70鹼基配對，對幾乎所有物種的tRNAAla而言，G3:U70都是唯一且不可或缺的識別決定基；如果改變tRNAAla的G3:U70，這個tRNA就無法被alanyl-tRNA synthetase (AlaRS)辨認及胺醯化；相反地，如果只是改變tRNAAla的反密碼，並不影響其胺醯化效率。蛋白質序列比對及三級結構分析顯示：AlaRS的tRNA結合位置上有二個高度保留的胺基酸(Asp及Asn)，這二個胺基酸專門負責辨認G3:U70。令人驚訝的是人類粒線體AlaRS (AlaRSm或AARS2)的相對應位置上並非Asp及Asn，更令人驚訝的是人類粒線體tRNAAla (tRNAmAla)上並沒有G3:U70。我們初步的實驗結果顯示：人類粒線體AlaRS辨認的識別決定基並非G3:U70。另外，最近有研究報告指稱人類粒線體AlaRS突變會引起嬰兒心肌症，但是詳細機制尚不清楚。為了解開這些謎團，我們擬定了一個三年期的研究計畫，重點包括：(1)研究人類粒線體AlaRS如何辨認非典型tRNAAla (沒有G3:U70)；(2)研究人類粒線體AlaRS突變為何會導致嬰兒心肌症。 ;Aminoacyl-tRNA synthetases (aaRSs) are a family of translation enzymes, each of which catalyzes the attachment of a specific amino acid to its cognate tRNAs. Each tRNA is recognized by its cognate aaRS through a specific set of “identity elements”, which often reside in the acceptor stem and anticodon. These identity elements are highly conserved throughout evolution. For example, almost all known alanine tRNA (tRNAAla) isoacceptors contain a G3:U70 wobble base pair in the acceptor stem that identifies tRNAAla for aminoacylation with alanine. Transfer of this GU base pair into a non-alanine tRNA efficiently converts it into an alanine acceptor. On the contrary, mutation of the anticodon of tRNAAla does not compromise its identity as an alanine acceptor. Sequence and structural studies show that two highly conserved amino acid residues, Asp and Asn, in the tRNA-binding domain of alanyl-tRNA synthetase (AlaRS) are responsible for recognition of the canonical identity elements G3:U70. Surprisingly, these two amino acid residues are absent from human mitochondrial AlaRS (AlaRSm or AARS2). Even more surprising was the finding that human mitochondrial tRNAAla (tRNAmAla) lacks G3:U70. As a result, AARS2 failed to charge tRNAAla with G3:U70 (unpublished data). This raised the question of how AARS2 recognizes its cognate tRNA. Moreover, a recent study showed that a homozygous mutation or compound heterogeneous mutations in human AARS2 cause infantile mitochondrial cardiomyopathy. However, the molecular mechanism underlying this disease is still elusive. The proposal presented herein elaborates a three-year project, in which we aim (1) to study how human AARS2 recognizes a noncanonical tRNAAla (without G3:U70), and (2) to study how human AARS2 mutations cause infantile mitochondrial cardiomyopathy (CM).
