YibK is the 2′-O-methyltransferase TrmL that modifies the wobble nucleotide in Escherichia coli tRNALeu isoacceptors
YibK is the 2′-O-methyltransferase TrmL that modifies the wobble nucleotide in Escherichia coli tRNALeu isoacceptors
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DOI：
10.1261/rna.2245910
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发表时间：
2010-11-01
期刊：
影响因子：
4.5
通讯作者：
Armengod, M-Eugenia
中科院分区：
文献类型：
--
作者：
Benitez-Paez, Alfonso;Villarroya, Magda;Armengod, M-Eugenia
转移RNA是活细胞中修饰最密集的核酸分子。在大肠杆菌中，已对 30 多种核苷修饰进行了表征，从甲基化和假尿苷化到需要多个酶促步骤的更复杂的添加。大多数修饰酶已被鉴定，但一些值得注意的例外包括 2'-O-甲基转移酶，其将两个亮氨酰同工受体 tRNA(CmAA)(Leu) 和 tRNA(cmnm5UmAA)(Leu) 中核苷酸 34 摆动位置处的核糖甲基化。在这里，我们使用比较基因组学方法来发现缺失酶的候选大肠杆菌基因。通过质谱分析来自候选基因无效突变体的转移 RNA，结果表明 yibK 失活导致 tRNA(CmAA)(Leu) 和 tRNA(cmnm5UmAA)(Leu) 中 34 位 2'-O-甲基化丢失。 YibK 甲基化的缺失会降低密码子-摆动碱基相互作用的效率，如琥珀抑制子supP系统所证明的那样。 yibK 失活对稳态生长速率没有可检测到的影响，尽管在多轮混合群体生长实验中注意到明显的缺点，表明从稳定期恢复的能力受到损害。通过与 yibK 的重组拷贝互补，甲基化可在体内恢复。尽管 YibK 是最小的 α/β 结蛋白之一，但它独立催化从 S-腺苷-L-甲硫氨酸到摆动核苷酸的 2'-OH 的甲基转移； YibK 识别该靶标需要 34 位上的吡啶和 37 位上的 N-6-(异戊烯基)-2-甲硫腺苷。 YibK 是最后剩下的待鉴定的大肠杆菌 tRNA 修饰酶之一，现已重命名为 TrmL。
Transfer RNAs are the most densely modified nucleic acid molecules in living cells. In Escherichia coli, more than 30 nucleoside modifications have been characterized, ranging from methylations and pseudouridylations to more complex additions that require multiple enzymatic steps. Most of the modifying enzymes have been identified, although a few notable exceptions include the 2'-O-methyltransferase(s) that methylate the ribose at the nucleotide 34 wobble position in the two leucyl isoacceptors tRNA(CmAA)(Leu) and tRNA(cmnm5UmAA)(Leu). Here, we have used a comparative genomics approach to uncover candidate E. coli genes for the missing enzyme(s). Transfer RNAs from null mutants for candidate genes were analyzed by mass spectrometry and revealed that inactivation of yibK leads to loss of 2'-O-methylation at position 34 in both tRNA(CmAA)(Leu) and tRNA(cmnm5UmAA)(Leu). Loss of YibK methylation reduces the efficiency of codon-wobble base interaction, as demonstrated in an amber suppressor supP system. Inactivation of yibK had no detectable effect on steady-state growth rate, although a distinct disadvantage was noted in multiple-round, mixed-population growth experiments, suggesting that the ability to recover from the stationary phase was impaired. Methylation is restored in vivo by complementing with a recombinant copy of yibK. Despite being one of the smallest characterized alpha/beta knot proteins, YibK independently catalyzes the methyl transfer from S-adenosyl-L-methionine to the 2'-OH of the wobble nucleotide; YibK recognition of this target requires a pyridine at position 34 and N-6-(isopentenyl)-2-methylthioadenosine at position 37. YibK is one of the last remaining E. coli tRNA modification enzymes to be identified and is now renamed TrmL.