Isolation and characterization of the human tRNA-(NIG37) methyltransferase (TRM5) and comparison to the Escherichia coli TrmD protein
Isolation and characterization of the human tRNA-(NIG37) methyltransferase (TRM5) and comparison to the Escherichia coli TrmD protein
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DOI：
10.1021/bi049671q
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发表时间：
2004-07-20
期刊：
影响因子：
2.9
通讯作者：
Holmes, WM
中科院分区：
文献类型：
--
作者：
Brulé, H;Elliott, M;Holmes, WM
已克隆了人TRM 5 cDNA，并产生重组tRNA-N（1）G37甲基转移酶。重组酶利用S-腺苷甲硫氨酸甲基化选定tRNA转录物中鸟苷37（G37）的N1位置。比较大肠杆菌TrmD和人TRM 5重组酶之间的甲基化反应的RNA序列和结构的影响。无论位置36处的核苷酸的性质如何，通过TRM 5的G37甲基化都会发生。与TrmD不同，TRM 5也使37位的肌苷甲基化，TrmD优先识别G36 pG 37基序，而不使肌苷甲基化。关于TrmD的新证据表明，与一些tRNA物种，在位置36的A也被认可。TRM 5酶对tRNA-蛋白质三级相互作用的细微变化敏感，导致活性丧失。TrmD酶对tRNA-蛋白质三级相互作用的改变更耐受，只要存在核心tRNA结构和G36 pG 37。TRM 5酶对镁离子没有绝对需求，而TrmD需要镁来表达活性。TRM 5对底物表现出高得多的亲和力，tRNA的K-m值为纳摩尔。TrmD对tRNA的Km值在微摩尔范围内。重组TRM 5似乎作为一个60 - 772 Da的单体，而重组TrmD作为一个同源二聚体的30 - 586 Da亚基的功能。对人类TRM 5基因组基因座（KIAA 1393）的生物信息学分析已经在真核生物和古细菌中鉴定出TRM 5同源物;然而，在包括TrmD基因在内的任何原核生物中均未鉴定出显著同源的区域。
A human TRM5 cDNA has been cloned and recombinant tRNA-N(1)G37 methyltransferase was produced. The recombinant enzyme methylates the N1 position of guanosine 37 (G37) in selected tRNA transcripts utilizing S-adenosyl methionine. The effects of RNA sequence and structure on the methylation reaction in comparison between the Escherichia coli TrmD and human TRM5 recombinant enzymes are presented. G37-methylation by TRM5 occurs regardless of the nature of the nucleotide at position 36. TRM5 also methylates inosine at position 37 unlike TrmD, which recognizes the G36pG37 motif preferentially and does not methylate inosine. New evidence is presented concerning TrmD showing that with some tRNA species, A at position 36 is also recognized. The TRM5 enzyme is sensitive to subtle changes in the tRNA-protein tertiary interaction leading to loss of activity. The TrmD enzyme is more tolerant of alterations in tRNA-protein tertiary interactions as long as the core tRNA structure and the G36pG37 are present. The TRM5 enzyme does not have an absolute requirement for magnesium ions, whereas TrmD requires magnesium to express activity. TRM5 demonstrates much higher affinity for substrates with K-m values for tRNA that are nanomolar. TrmD has K-m values for tRNA in the micromolar range. Recombinant TRM5 appears to function as a 60 772 Da monomer, while recombinant TrmD functions as a homodimer of 30 586 Da subunits. Bioinformatic analysis of the human TRM5 genomic locus (KIAA 1393) have identified TRM5 homologues in eukaryotes and archaea; however, no significantly homologous regions were identified in any prokaryotes including the TrmD gene.