Identification of determinants for tRNA substrate recognition by Escherichia coli C/U34 2'-O-methyltransferase.
Identification of determinants for tRNA substrate recognition by Escherichia coli C/U34 2'-O-methyltransferase.
复制标题
DOI：
10.1080/15476286.2015.1050576
复制
发表时间：
2015
期刊：
影响因子：
4.1
通讯作者：
Wang ED
中科院分区：
文献类型：
--
作者：
Zhou M;Long T;Fang ZP;Zhou XL;Liu RJ;Wang ED
转录后修饰为tRNA带来了化学多样性，特别是在反密码子茎环（ASL）的34和37位。TrmL是原核甲基转移酶，催化甲基从S-腺苷-L-甲硫氨酸转移至tRNALeuCAA和tRNALeuUAA异源受体的摆动碱基。这种Cm 34/Um 34修饰影响密码子-反密码子相互作用，并且对翻译保真度至关重要。TrmL催化的2′-O-甲基化需要其同源二聚化;然而，对TrmL识别tRNA的机制的理解仍然是难以捉摸的。在目前的研究中，通过测量tRNA甲基化的TrmL和进行动力学分析的tRNA突变体，我们发现，TrmL表现出微调tRNA底物识别机制。具有2个碱基对延伸的反密码子茎环微螺旋是EcTrmL甲基化的最小底物。A35是TrmL识别的关键残基，而A36-A37-A38是重要的，通过与TrmL的直接相互作用或由于在A37处的预先异戊烯化（i6）的必要性。此外，TrmL仅甲基化嘧啶，而不是在摆动位置的嘌呤残基，并且2′-O-甲基化依赖于在位置37处的先前N6-异戊烯基腺苷修饰。
Post-transcriptional modifications bring chemical diversity to tRNAs, especially at positions 34 and 37 of the anticodon stem-loop (ASL). TrmL is the prokaryotic methyltransferase that catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the wobble base of tRNALeuCAA and tRNALeuUAA isoacceptors. This Cm34/Um34 modification affects codon-anticodon interactions and is essential for translational fidelity. TrmL-catalyzed 2′-O-methylation requires its homodimerization; however, understanding of the tRNA recognition mechanism by TrmL remains elusive. In the current study, by measuring tRNA methylation by TrmL and performing kinetic analysis of tRNA mutants, we found that TrmL exhibits a fine-tuned tRNA substrate recognition mechanism. Anticodon stem-loop minihelices with an extension of 2 base pairs are the minimal substrate for EcTrmL methylation. A35 is a key residue for TrmL recognition, while A36-A37-A38 are important either via direct interaction with TrmL or due to the necessity for prior isopentenylation (i6) at A37. In addition, TrmL only methylates pyrimidines but not purine residues at the wobble position, and the 2′-O-methylation relies on prior N6-isopentenyladenosine modification at position 37.