ACS Chem Biol. 2011 Nov 18;6(11):1182-7. doi: 10.1021/cb200240a. Epub 2011 Sep 8.
The yeast Saccharomyces cerevisiae contains two distinct nuclear glycyl-tRNA synthetase (GlyRS) genes, GRS1 and GRS2. GRS1 is dual functional in that possesses both cytoplasmic and mitochondrial activities, whereas GRS2 is pseudogene-like. GlyRS1 and GlyRS2 are highly similar on the whole but are distinguished by a lysine-rich insertion domain of 44 amino acid residues, present only in GlyRS1. We herein present evidence that whereas the insertion domain is dispensable for the complementary activity of GRS1in vivo, deletion of this domain from GlyRS1 reduced its aminoacylation activity by up to 9-fold. On the other hand, fusion of a constitutive ADH promoter to GRS2 failed to confer a functional phenotype to the gene, but further fusion of ARC1 (a yeast gene encoding a tRNA-binding protein, Arc1p) to this hybrid gene successfully rescued its activity. Most intriguingly, purified GlyRS2 retained a substantial level of aminoacylation activity. Fusion of Arc1p to this enzyme further enhanced its activity and stability. These findings highlight not only the structural integrity of the pseudogene-encoded enzyme but also the necessity of obtaining an auxiliary tRNA-binding domain for functioning of a yeast tRNA synthetase.
酿酒酵母含有两个不同的核甘氨酰-tRNA 合成酶（GlyRS）基因，GRS1 和 GRS2。GRS1 具有双重功能，既具有细胞质活性又具有线粒体活性，而 GRS2 则类似于假基因。GlyRS1 和 GlyRS2 在整体上非常相似，但在 GlyRS1 中存在一个由 44 个氨基酸残基组成的富含赖氨酸的插入结构域，这是它们的区别。我们在此证明，尽管插入结构域对于 GlyRS1 在体内的互补活性不是必需的，但从 GlyRS1 中删除该结构域会使其氨酰化活性降低多达 9 倍。另一方面，将组成型 ADH 启动子融合到 GRS2 上并不能赋予该基因功能性表型，但将 ARC1（一种编码 tRNA 结合蛋白的酵母基因，Arc1p）进一步融合到这个杂合基因上成功地挽救了其活性。最有趣的是，纯化的 GlyRS2 保留了相当水平的氨酰化活性。将 Arc1p 融合到该酶中进一步增强了其活性和稳定性。这些发现不仅强调了假基因编码酶的结构完整性，还强调了获得辅助 tRNA 结合结构域对于酵母 tRNA 合成酶功能的必要性。
