Modified uridines with c5-methylene substituents at the first position of the tRNA anticodon stabilize U•G wobble pairing during decoding
Modified uridines with c5-methylene substituents at the first position of the tRNA anticodon stabilize U•G wobble pairing during decoding
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DOI：
10.1074/jbc.m800233200
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发表时间：
2008-07-04
影响因子：
4.8
通讯作者：
Suzuki, Tsutomu
中科院分区：
文献类型：
--
作者：
Kurata, Shinya;Weixlbaumer, Albert;Suzuki, Tsutomu
转运RNA（tRNA）反密码子第一位（摆动位）的转录后修饰参与遗传密码的精确解码。为了解码以嘌呤（R）结尾的密码子（即NNR），tRNAs经常在摆动位使用5 - 甲基尿苷衍生物（xm(5)U）。然而，xm(5)U的C5取代基在密码子识别中的功能特性仍然难以捉摸。我们先前发现，从线粒体脑肌病伴高乳酸血症和卒中样发作（MELAS）患者中分离出的具有致病性点突变的线粒体tRNAs(Leu(UUR))缺乏5 - 牛磺甲基尿苷（tau m(5)U）修饰，并导致解码缺陷。在此，我们构建了在摆动位有或没有xm(5)U修饰的大肠杆菌tRNAs(Leu(UUR))，并通过体外翻译以及A位点tRNA结合来测量它们的解码活性。此外，通过使用具有连续AGR密码子的报告基因构建体进行脉冲标记，检测了在修饰酶敲除菌株（ΔmnmE）中缺乏mnm(5)U修饰的tRNA(Arg)的解码特性。我们的结果表明，xm(5)U修饰通过稳定摆动位的U·G配对在解码NNG密码子中起关键作用。含有tau m(5)U的反密码子茎环与核糖体A位点的UUA或UUG密码子相互作用的晶体结构显示，m(5)U·G碱基对不具有经典的U·G摆动几何结构。这些结构有助于解释tau m(5)U修饰如何使UUG密码子能够有效解码。
Post-transcriptional modifications at the first ( wobble) position of the tRNA anticodon participate in precise decoding of the genetic code. To decode codons that end in a purine (R) (i.e. NNR), tRNAs frequently utilize 5-methyluridine derivatives (xm(5)U) at the wobble position. However, the functional properties of the C5-substituents of xm(5)U in codon recognition remain elusive. We previously found that mitochondrial tRNAs(Leu(UUR)) with pathogenic point mutations isolated from MELAS ( mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) patients lacked the 5-taurinomethyluridine (tau m(5)U) modification and caused a decoding defect. Here, we constructed Escherichia coli tRNAs(Leu(UUR)) with or without xm(5)U modifications at the wobble position and measured their decoding activities in an in vitro translation as well as by A-site tRNA binding. In addition, the decoding properties of tRNA(Arg) lacking mnm(5)U modification in a knock-out strain of the modifying enzyme (Delta mnmE) were examined by pulse labeling using reporter constructs with consecutive AGR codons. Our results demonstrate that the xm(5)U modification plays a critical role in decoding NNG codons by stabilizing U center dot G pairing at the wobble position. Crystal structures of an anticodon stem-loop containing tau m(5)U interacting with a UUA or UUG codon at the ribosomal A-site revealed that the m(5)U center dot G base pair does not have classical U center dot G wobble geometry. These structures provide help to explain how the tau m(5)U modification enables efficient decoding of UUG codons.