The dynamic NMR structure of the T psi C-loop: implications for the specificity of tRNA methylation.
The dynamic NMR structure of the T psi C-loop: implications for the specificity of tRNA methylation.
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T psi C 环的动态 NMR 结构：对 tRNA 甲基化特异性的影响。
DOI：
10.1023/a:1018618606857
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发表时间：
1997
影响因子：
2.7
通讯作者：
Schmitz,U
中科院分区：
文献类型：
--
作者：
Yao,LJ;James,TL;Kealey,JT;Santi,DV;Schmitz,U
tRNA (m5U54)-甲基转移酶 (RUMT) 催化大肠杆菌中所有转移 RNA 的 TΨC 环中尿苷 54 的 S-腺苷甲硫氨酸依赖性甲基化，形成 54-核糖胸腺嘧啶残基。然而，在所有 tRNA 结构中，残基 54 被完全掩埋，并且出现了 RUMT 如何获得甲基化位点的问题。由 TΨ 环的核苷酸 49-65 组成的 17 聚体 RNA 发夹是 RUMT 的底物。同核 NMR 方法与限制分子动力学 (MD) 方法相结合，用于确定 17 聚体 T 臂片段的溶液结构。发夹环表现出增强的柔韧性，这使得传统的NMR平均结构与分子主要以一种主要构象存在的更常见的情况相比不太有用。然而，当采用更软的细化方法（例如具有时间平均约束的MD）时，可以更好地满足循环中的冲突约束。 T 形臂的动态结构被表示为 10 个时间簇的集合。在这一切之中，U54彻底暴露无遗。 TΨ 环在溶液中的灵活性以及 RUMT 与 TΨC 环和 tRNA 的广泛结合研究表明 RUMT/tRNA 识别的特异性与 tRNA 三级结构元件相关。对于甲基化，RUMT 只需要破坏 D 环和 T 环之间的三级相互作用，导致 T 臂结构熔化并使 U54 可用于甲基化。
tRNA (m5U54)-methyltransferase (RUMT) catalyzes the S-adenosylmethionine-dependentmethylation of uridine-54 in the TΨC-loop of all transfer RNAs in E. coli to form the 54-ribosylthymine residue. However, in all tRNA structures, residue 54 is completely buried andthe question arises as to how RUMT gains access to the methylation site. A 17-mer RNAhairpin consisting of nucleotides 49–65 of the TΨ-loop is a substrate for RUMT.Homonuclear NMR methods in conjunction with restrained molecular dynamics (MD)methods were used to determine the solution structure of the 17-mer T-arm fragment. Theloop of the hairpin exhibits enhanced flexibility which renders the conventional NMR averagestructure less useful compared to the more commonly found situation where a molecule existsin predominantly one major conformation. However, when resorting to softer refinementmethods such as MD with time-averaged restraints, the conflicting restraints in the loop canbe satisfied much better. The dynamic structure of the T-arm is represented as an ensembleof 10 time-clusters. In all of these, U54 is completely exposed. The flexibility of the TΨ-loop in solution in conjunction with extensive binding studies of RUMT with the TΨC-loop and tRNA suggest that the specificity of the RUMT/tRNA recognition is associated withtRNA tertiary structure elements. For the methylation, RUMT would simply have to breakthe tertiary interactions between the D- and T-loops, leading to a melting of the T-armstructure and making U54 available for methylation.