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酵母苯丙氨酸转运核糖核酸(tRNA(Phe))修饰核苷在核糖体结合中的作用

Role of modified nucleosides of yeast tRNA(Phe) in ribosomal binding.

作者信息

Ashraf S S, Guenther R H, Ansari G, Malkiewicz A, Sochacka E, Agris P F

机构信息

Novalon Pharmaceutical Corporation, Durham, NC 27703, USA.

出版信息

Cell Biochem Biophys. 2000;33(3):241-52. doi: 10.1385/cbb:33:3:241.

Abstract

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 (Gm34) 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 Gm34 with 16S rRNA C1400.

摘要

天然存在的核苷修饰是转运RNA(tRNA)的一个固有特征,并且与密码子识别的效率和准确性有关。研究了酵母tRNA(Phe)反密码子结构域中修饰核苷的结构和功能贡献。修饰核苷被位点选择性地单独或组合掺入到十七聚体反密码子茎环结构域(ASL(Phe))中。茎修饰,即5-甲基胞嘧啶,提高了RNA的热稳定性,但对核糖体结合有有害影响。相反,环修饰,即1-甲基鸟苷,增强了核糖体结合,但显著降低了热稳定性。当存在多种修饰时,整体ASL稳定性主要是茎和环各自贡献的结果。修饰对核糖体结合的影响不能从热力学贡献或在茎或环中的位置预测。当ASL中有4/5个修饰时,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与密码子阅读的准确性有关,而不是与该tRNA对核糖体P位点的亲和力有关。此外,我们使用位点选择性掺入特定核苷修饰的方法,确定了摆动位置34处鸟苷的2'-O-甲基化(Gm34)是导致酵母tRNA(Phe)核糖体足迹分析时大肠杆菌16S rRNA中C1400化学反应性显著增强的原因。因此,tRNA(Phe)与核糖体的有效结合是反密码子茎稳定性以及反密码子环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Gm34与16S rRNA C1400的相互作用。

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