Role of modified nucleosides of yeast tRNAPhe in ribosomal binding
Role of modified nucleosides of yeast tRNAPhe in ribosomal binding
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DOI：
10.1385/cbb:33:3:241
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发表时间：
2000-01-01
影响因子：
2.6
通讯作者：
Agris, PF
中科院分区：
文献类型：
--
作者：
Ashraf, SS;Guenther, RH;Agris, PF
天然发生的核苷修饰是转移 RNA (tRNA) 的固有特征，并且与密码子识别的效率和准确性有关。检查了酵母 tRNA（Phe）反密码子结构域中修饰核苷的结构和功能贡献。修饰的核苷被位点选择性地单独或组合地掺入十七聚体反密码子茎和环结构域（ASL（Phe））。茎修饰 5-甲基胞苷提高了 RNA 的热稳定性，但对核糖体结合产生有害影响。相反，环修饰 1-甲基鸟苷增强了核糖体结合，但显着降低了热稳定性。由于存在多种修改，全局 ASL 稳定性主要是个体对茎的贡献加上对环的贡献的结果。修饰对核糖体结合的影响无法从热力学贡献或茎或环中的位置来预测。 ASL 进行 4/5 修饰后，核糖体结合与未修饰的 ASL 相当。因此，酵母 tRNA(Phe) 反密码子结构域的修饰可能更多地与密码子读取的准确性有关，而不是与该 tRNA 对核糖体 P 位点的亲和力有关。此外，我们还使用特定核苷修饰的位点选择性掺入方法来鉴定摆动位置 34 (Gm(34)) 处鸟苷的 2'O-甲基化，该甲基化是在酵母 tRNA(Phe) 核糖体足迹分析中导致大肠杆菌 16S rRNA 中 C1400 化学反应性特征性增强的原因。因此，tRNA（Phe）的有效核糖体结合是反密码子茎稳定性和反密码子环的正确结构和动力学的结合。 tRNA 与核糖体 P 位点的正确结合可能包括 Gm(34) 与 16S rRNA C1400 的相互作用。
Naturally occurring nucleoside modifications are an intrinsic feature of transfer RNA (tRNA), and have been implicated in the efficiency, as well as accuracy-of codon recognition. The structural and functional contributions of the modified nucleosides in the yeast tRNA(Phe) anticodon domain were examined. Modified nucleosides were site-selectively incorporated, individually and in combinations, into the heptadecamer anticodon stem and loop domain, (ASL(Phe)). The stem modification, 5-methylcytidine, improved RNA thermal stability, but had a deleterious effect on ribosomal binding. In contrast, the loop modification, 1-methylguanosine, enhanced ribosome binding, but dramatically decreased thermal stability. With multiple modifications present, the global ASL stability was mostly the result of the individual contributions to the stem plus that to the loop. The effect of modification on ribosomal binding was not predictable from thermodynamic contributions or location in the stem or loop. With 4/5 modifications in the ASL, ribosomal binding was comparable to that of the unmodified ASL. Therefore, modifications of the yeast tRNA(Phe) anticodon domain may have more to do with accuracy of codon reading than with affinity of this tRNA for the ribosomal P-site. in addition, we have used the approach of site-selective incorporation of specific nucleoside modifications to identify 2'O-methylation of guanosine at wobble position 34 (Gm(34)) as being responsible for the characteristically enhanced chemical reactivity of C1400 in Escherichia coli 16S rRNA upon ribosomal footprinting of yeast tRNA(Phe). Thus, effective ribosome binding of tRNA(Phe) is a combination of anticodon stem stability and the correct architecture and dynamics of the anticodon loop. Correct tRNA binding to the ribosomal P-site probably includes interaction of Gm(34) with 16S rRNA C1400.