Gatti D L, Tzagoloff A
Department of Biological Sciences, Columbia University, New York, NY 10027.
J Mol Biol. 1991 Apr 5;218(3):557-68. doi: 10.1016/0022-2836(91)90701-7.
A yeast nuclear gene, designated MSK1, has been selected from a yeast genomic library by transformation of a respiratory deficient mutant impaired in acylation of mitochondrial lysine tRNA. This gene confers a respiratory competent phenotype and restores the mutant's ability to acylate the mitochondrial lysine tRNA. The amino acid sequence of the protein encoded by MSK1 is homologous to yeast cytoplasmic lysyl-tRNA synthetase and to the product of the herC gene, which has recently been suggested to code for the Escherichia coli enzyme. These observations indicate that MSK1 codes for the lysyl-tRNA synthetase of yeast mitochondria. Several regions of high primary sequence conservation have been identified in the bacterial and yeast lysyl-tRNA synthetases. These domains are also present in the aspartyl- and asparaginyl-tRNA synthetases, further confirming the notion that all three present-day enzymes originated from a common ancestral gene. The most conserved domain, located near the carboxyl terminal ends of this group of synthetases is characterized by a cluster of glycines and is also highly homologous to the carboxyl-terminal region of the E. coli ammonia-dependent asparagine synthetase. A catalytic function of the carboxyl terminal domain is indicated by in vitro mutagenesis of the yeast mitochondrial lysyl-tRNA synthetase. Replacement of any one of three glycine residues by alanine and in one case by aspartic acid completely suppresses the activity of the enzymes, as evidenced by the inability of the mutant genes to complement an msk1 mutant, even when present in high copy. Other mutations result in partial loss of activity. Only one glycine replacement affects the stability of the protein in vivo. The observed presence of a homologous domain in asparagine synthetase, which, like the aminoacyl-tRNA synthetases, catalyzes the formation of an aminoacyladenylate, suggests that the glycine-rich sequence is part of a catalytic site involved in binding of ATP and of the aminoacyladenylate intermediate.
通过转化线粒体赖氨酸tRNA酰化受损的呼吸缺陷型突变体，从酵母基因组文库中筛选出一个名为MSK1的酵母核基因。该基因赋予呼吸正常的表型，并恢复突变体酰化线粒体赖氨酸tRNA的能力。MSK1编码的蛋白质的氨基酸序列与酵母细胞质赖氨酰-tRNA合成酶以及herC基因的产物同源，最近有人提出herC基因编码大肠杆菌的这种酶。这些观察结果表明，MSK1编码酵母线粒体的赖氨酰-tRNA合成酶。在细菌和酵母赖氨酰-tRNA合成酶中已鉴定出几个高度保守的一级序列区域。这些结构域也存在于天冬氨酰-和天冬酰胺-tRNA合成酶中，进一步证实了当今所有这三种酶都起源于一个共同祖先基因的观点。最保守的结构域位于这组合成酶的羧基末端附近，其特征是有一簇甘氨酸，并且与大肠杆菌氨依赖性天冬酰胺合成酶的羧基末端区域也高度同源。酵母线粒体赖氨酰-tRNA合成酶的体外诱变表明了羧基末端结构域的催化功能。将三个甘氨酸残基中的任何一个替换为丙氨酸，在一种情况下替换为天冬氨酸，会完全抑制酶的活性，这可通过突变基因无法互补msk1突变体来证明，即使以高拷贝存在时也是如此。其他突变导致活性部分丧失。只有一个甘氨酸替换影响蛋白质在体内的稳定性。在天冬酰胺合成酶中观察到存在同源结构域，天冬酰胺合成酶与氨酰-tRNA合成酶一样，催化氨酰腺苷酸的形成，这表明富含甘氨酸的序列是参与ATP和氨酰腺苷酸中间体结合的催化位点的一部分。
