Biogenesis and growth phase-dependent alteration of 5-methoxycarbonylmethoxyuridine in tRNA anticodons.
Biogenesis and growth phase-dependent alteration of 5-methoxycarbonylmethoxyuridine in tRNA anticodons.
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DOI：
10.1093/nar/gkv1470
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发表时间：
2016-01-29
影响因子：
14.9
通讯作者：
Suzuki T
中科院分区：
文献类型：
--
作者：
Sakai Y;Miyauchi K;Kimura S;Suzuki T
在tRNA的反密码子第一（摆动）位置的转录后修饰在遗传密码的精确解码中起着关键作用。5-羧基甲氧基尿苷（cmo 5 U）及其甲酯衍生物5-甲氧基羰基甲氧基尿苷（mcmo 5 U）是在来自革兰氏阴性菌的几种tRNA的反密码子摆动位置发现的修饰核苷。cmo 5 U和mcmo 5 U促进与密码子第三位的鸟苷和嘧啶的非沃森-克里克碱基配对，从而扩展解码能力。通过对单个tRNA的质谱分析和对大肠杆菌总RNA的鸟枪法分析，我们确定mcmo 5 U是tRNAAla 1、tRNASer 1、tRNAPro 3和tRNAThr 4中的主要修饰;相比之下，cmo 5 U主要存在于tRNALeu 3和tRNAVal 1中。此外，我们发现5-甲氧羰基甲氧基-2 ′-O-甲基尿苷（mcmo 5 Um）作为一种新的，但在tRNASer 1的微小修饰。tRNAPro 3中mcmo 5 U的末端甲基化频率在细胞生长的早期对数期较低（约30%），随着生长的进行逐渐增加，并在晚期对数期和稳定期达到接近100%。我们发现了CmoM（以前称为SmtA），这是一种AdoMet依赖性甲基转移酶，可将cmo 5 U甲基化形成mcmo 5 U。基于+1移码构建体的荧光素酶报告基因测定显示，mcmo 5 U的末端甲基化有助于tRNAAla 1的解码能力。
Post-transcriptional modifications at the anticodon first (wobble) position of tRNA play critical roles in precise decoding of genetic codes. 5-carboxymethoxyuridine (cmo5U) and its methyl ester derivative 5-methoxycarbonylmethoxyuridine (mcmo5U) are modified nucleosides found at the anticodon wobble position in several tRNAs from Gram-negative bacteria. cmo5U and mcmo5U facilitate non-Watson–Crick base pairing with guanosine and pyrimidines at the third positions of codons, thereby expanding decoding capabilities. By mass spectrometric analyses of individual tRNAs and a shotgun approach of total RNA from Escherichia coli, we identified mcmo5U as a major modification in tRNAAla1, tRNASer1, tRNAPro3 and tRNAThr4; by contrast, cmo5U was present primarily in tRNALeu3 and tRNAVal1. In addition, we discovered 5-methoxycarbonylmethoxy-2′-O-methyluridine (mcmo5Um) as a novel but minor modification in tRNASer1. Terminal methylation frequency of mcmo5U in tRNAPro3 was low (≈30%) in the early log phase of cell growth, gradually increased as growth proceeded and reached nearly 100% in late log and stationary phases. We identified CmoM (previously known as SmtA), an AdoMet-dependent methyltransferase that methylates cmo5U to form mcmo5U. A luciferase reporter assay based on a +1 frameshift construct revealed that terminal methylation of mcmo5U contributes to the decoding ability of tRNAAla1.