Identification of the TRM2 gene encoding the tRNA(m5U54) methyltransferase of Saccharomyces cerevisiae
Identification of the TRM2 gene encoding the tRNA(m5U54) methyltransferase of Saccharomyces cerevisiae
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DOI：
10.1017/s1355838200992422
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发表时间：
2000-06-01
期刊：
影响因子：
4.5
通讯作者：
Byström, AS
中科院分区：
文献类型：
--
作者：
Nordlund, ME;Johansson, JOM;Byström, AS
在54位存在5-甲基尿苷（m（5）U）是大多数细菌和真核生物延伸子tRNA的普遍特征。在这项研究中，我们已经确定并表征了编码tRNA（m5 U 54）甲基转移酶的TRM 2基因，该基因负责在酿酒酵母中形成这种修饰的核苷。从TRM 2破坏的酵母菌株中分离的转移RNA不含m5 U核苷。此外，谷胱甘肽S-转移酶（GST）标记的重组体，Trm 2 p，在大肠杆菌中表达的tRNA（m5 U 54）甲基转移酶活性，使用从trm 2突变株分离的tRNA作为底物，但不是从TRM 2野生型菌株分离的tRNA。与在大肠杆菌中发现的tRNA（m5 U 54）甲基转移酶编码基因trmA（+）相反，在大肠杆菌中，TRM 2基因对细胞活力不是必需的，缺失菌株没有明显的表型。令人惊讶的是，我们发现TRM 2基因先前被鉴定为RNC 1/NUD 1基因，据信编码yNucR内切核酸酶。yNucR内切核酸酶的表达和活性依赖于RAD 52基因，并且不响应于RNC 1/NUD 1基因的增加的基因剂量。相反，我们发现trm 2-lacZ融合体的表达和tRNA（m（5）U（54））甲基转移酶的活性不受RAD 52基因的调节，并且确实对TRM 2（RNC 1/NUD 1）基因的增加的基因剂量有反应。此外，没有与GST-Trm 2重组蛋白相关的核酸酶活性。已报道纯化的yNucR内切-外切核酸酶具有NH 2-D-E-K-N-L基序，其在Trm 2 p中未发现。因此，我们认为yNucR内切核酸酶由TRM 2以外的基因编码。
The presence of 5-methyluridine (m(5)U) at position 54 is a ubiquitous feature of most bacterial and eukaryotic elongator tRNAs. In this study, we have identified and characterized the TRM2 gene that encodes the tRNA(m5U54)methyltransferase, responsible for the formation of this modified nucleoside in Saccharomyces cerevisiae. Transfer RNA isolated from TRM2-disrupted yeast strains does not contain the m5U,, nucleoside. Moreover, a glutathione S-transferase (GST) tagged recombinant, Trm2p, expressed in Escherichia coli displayed tRNA(m5U54)methyltransferase activity using as substrate tRNA isolated from a trm2 mutant strain, but not tRNA isolated from a TRM2 wild-type strain. In contrast to what is found for the tRNA(m5U54) methyltransferase encoding gene trmA(+) in E. coli, the TRM2 gene is not essential for cell Viability and a deletion strain shows no obvious phenotype. Surprisingly, we found that the TRM2 gene was previously identified as the RNC1/NUD1 gene, believed to encode the yNucR endo-exonuclease. The expression and activity of the yNucR endo-exonuclease is dependent on the RAD52 gene, and does not respond to increased gene dosage of the RNC1/NUD1 gene. In contrast, we find that the expression of a trm2-lacZ fusion and the activity of the tRNA(m(5)U(54))methyltransferase is not regulated by the RAD52 gene and does respond on increased gene dosage of the TRM2 (RNC1/NUD1) gene. Furthermore, there was no nuclease activity associated with a GST-Trm2 recombinant protein. The purified yNucR endo-exonuclease has been reported to have an NH2-D-E-K-N-L motif, which is not found in the Trm2p. Therefore, we suggest that the yNucR endo-exonuclease is encoded by a gene other than TRM2.