Liu J, Zhou G Q, Stråby K B
Department of Microbiology, Umeå University, S-901 87 Umeå, Sweden.
Gene. 1999 Jan 8;226(1):73-81. doi: 10.1016/s0378-1119(98)00550-2.
It has been estimated that eukaryotes carry more than 50 genes for tRNA modifying enzymes. Of the few so far identified most come from yeast, a lower eukaryote. In Saccharomyces cerevisiae, the TRM1 gene is a nuclear gene encoding the tRNA(m2/ 2G(26))dimethyltransferase, which catalyses the formation of the N2, N2-dimethylguanosine at position 26 in tRNA. We have isolated and characterized the corresponding gene ZC376.5 in Caenorhabditis elegans. Via RTPCR the cDNA sequence of the full length ZC376.5 has now been cloned, expressed in Escherichia coli and demonstrated to encode a tRNA(m2/2G(26))dimethyltransferase that produces dimethyl-G26 in vivo and in vitro with tRNA from yeast and bacteria as substrates. This is the first example of a complete gene sequence coding for a tRNA modifying enzyme from a multicellular organism. A point mutation in exon IV in the C. elegans genome sequence coding for the tRNA(m2/2G(26))methyltransferase that substituted arginine246 for glycine eliminated the modification activity. Exchanging the corresponding lysine residue in the yeast Trm1p for alanine caused a severe loss of activity, indicating that the identity of the amino acid at this position is important for enzyme activity.
据估计，真核生物携带超过50个用于tRNA修饰酶的基因。到目前为止，已鉴定出的少数基因大多来自低等真核生物酵母。在酿酒酵母中，TRM1基因是一个核基因，编码tRNA(m2/ 2G(26))二甲基转移酶，该酶催化tRNA第26位的N2,N2-二甲基鸟苷的形成。我们已经在秀丽隐杆线虫中分离并鉴定了相应的基因ZC376.5。通过RTPCR，现已克隆出全长ZC376.5的cDNA序列，在大肠杆菌中表达，并证明其编码一种tRNA(m2/2G(26))二甲基转移酶，该酶以酵母和细菌的tRNA为底物，在体内和体外产生二甲基-G26。这是来自多细胞生物的编码tRNA修饰酶的完整基因序列的首个实例。秀丽隐杆线虫基因组序列中编码tRNA(m2/2G(26))甲基转移酶的外显子IV中的一个点突变，将精氨酸246替换为甘氨酸，消除了修饰活性。将酵母Trm1p中相应的赖氨酸残基替换为丙氨酸导致活性严重丧失，表明该位置氨基酸的同一性对酶活性很重要。
