John T R, Ghosh M, Johnson J D
Department of Molecular Biology, University of Wyoming, Laramie 82071, USA.
Yeast. 1997 Jan;13(1):37-41. doi: 10.1002/(SICI)1097-0061(199701)13:1<37::AID-YEA55>3.0.CO;2-L.
The enzymes that aminoacylate tRNAs have been studied extensively and can be organized into two distinct classes based on signature sequences and the position of aminoacylation. The class I enzymes have canonical HIGH and KMSKS sequences as part of a Rossman fold nucleotide-binding site. The tryptophan-specific enzymes have been placed in class I based on analysis of the cognate genes from Escherichia coli, B. stearothermophilus, B. taurus, and Homo sapiens. An unidentified open reading frame (ORF) on Saccharomyces cerevisiae chromosome XV, HRE342, has 46% identity with the bovine tryptophanyl-tRNA synthetase and possesses the appropriate signature sequences. The predicted molecular weight of the putative HRE342 protein also closely matched the expected monomer size of the S. cerevisiae enzyme. The HRE342 ORF plus about 250 bp of 5' and 3' flanking sequence was amplified by polymerase chain reaction, cloned into a 2 mu based vector, and transformed into a host strain, S. cerevisiae JG369.3B. Nucleotide sequence analysis of the clone confirmed the presence of HRE342. Extracts from transformed yeast have a 30- to 100-fold increase in specific activity of the tryptophanyl-tRNA synthetase. An HRE342 locus in a diploid strain, PTY33XPTY44, was disrupted with a LEU2 insert. Sporulation and tetrad analysis of the HRE342::LEU2 strain demonstrated that HRE342 is an essential gene. We conclude that HRE342 is the S. cerevisiae gene encoding the cytoplasmic tryptophanyl-tRNA synthetase, WRS1. A search of the Saccharomyces Genome Database using amino acid sequences from other eukaryotic aminoacyl-tRNA synthetase suggests there is sufficient similarity to identify both class I and class II genes.
对氨酰化tRNA的酶已进行了广泛研究，根据特征序列和氨酰化位置可将其分为两个不同的类别。I类酶具有典型的HIGH和KMSKS序列，作为Rossman折叠核苷酸结合位点的一部分。基于对来自大肠杆菌、嗜热栖热菌、牛和人类的同源基因的分析，色氨酸特异性酶被归为I类。酿酒酵母第十五号染色体上一个未鉴定的开放阅读框（ORF）HRE342，与牛色氨酰 - tRNA合成酶有46%的同一性，并且拥有合适的特征序列。推测的HRE342蛋白的预测分子量也与酿酒酵母该酶预期的单体大小紧密匹配。通过聚合酶链反应扩增HRE342 ORF及其约250 bp的5'和3'侧翼序列，克隆到一个2μm的载体中，并转化到宿主菌株酿酒酵母JG369.3B中。对该克隆的核苷酸序列分析证实了HRE342的存在。来自转化酵母的提取物中色氨酰 - tRNA合成酶的比活性提高了30至100倍。在二倍体菌株PTY33XPTY44中，用LEU2插入片段破坏了HRE342位点。对HRE342::LEU2菌株的孢子形成和四分体分析表明，HRE342是一个必需基因。我们得出结论，HRE342是酿酒酵母中编码细胞质色氨酰 - tRNA合成酶WRS1的基因。利用其他真核氨酰 - tRNA合成酶的氨基酸序列在酿酒酵母基因组数据库中进行搜索，结果表明有足够的相似性来鉴定I类和II类基因。
