Liu M, Chu W C, Liu J C, Horowitz J
Department of Biochemistry and Biophysics, Iowa State University, Ames, IA 50011, USA.
Nucleic Acids Res. 1997 Dec 15;25(24):4883-90. doi: 10.1093/nar/25.24.4883.
Although the anticodon is the primary element in Escherichia coli tRNAValfor recognition by valyl-tRNA synthetase (ValRS), nucleotides in the acceptor stem and other parts of the tRNA modulate recognition. Study of the steady state aminoacylation kinetics of acceptor stem mutants of E.coli tRNAValdemonstrates that replacing any base pair in the acceptor helix with another Watson-Crick base pair has little effect on aminoacylation efficiency. The absence of essential recognition nucleotides in the acceptor helix was confirmed by converting E.coli tRNAAlaand yeast tRNAPhe, whose acceptor stem sequences differ significantly from that of tRNAVal, to efficient valine acceptors. This transformation requires, in addition to a valine anticodon, replacement of the G:U base pair in the acceptor stem of these tRNAs. Mutational analysis of tRNAValverifies that G:U base pairs in the acceptor helix act as negative determinants of synthetase recognition. Insertion of G:U in place of the conserved U4:A69 in tRNAValreduces the efficiency of aminoacylation, due largely to an increase in K m. A smaller but significant decline in aminoacylation efficiency occurs when G:U is located at position 3:70; lesser effects are observed for G:U at other positions in the acceptor helix. The negative effects of G:U base pairs are strongly correlated with changes in helix structure in the vicinity of position 4:69 as monitored by19F NMR spectroscopy of 5-fluorouracil-substituted tRNAVal. This suggests that maintaining regular A-type RNA helix geometry in the acceptor stem is important for proper recognition of tRNAValby valyl-tRNA synthetase.19F NMR also shows that formation of the tRNAVal-valyl-tRNA synthetase complex does not disrupt the first base pair in the acceptor stem, a result different from that reported for the tRNAGln-glutaminyl-tRNA synthetase complex.
尽管反密码子是大肠杆菌缬氨酰 - tRNA合成酶（ValRS）识别tRNAVal的主要元件，但tRNA受体茎及其他部位的核苷酸会调节这种识别。对大肠杆菌tRNAVal受体茎突变体的稳态氨酰化动力学研究表明，用另一个沃森 - 克里克碱基对替换受体螺旋中的任何碱基对，对氨酰化效率影响很小。通过将受体茎序列与tRNAVal显著不同的大肠杆菌tRNAAla和酵母tRNAPhe转化为有效的缬氨酸受体，证实了受体螺旋中不存在必需的识别核苷酸。这种转化除了需要缬氨酸反密码子外，还需要替换这些tRNA受体茎中的G:U碱基对。对tRNAVal的突变分析证实，受体螺旋中的G:U碱基对是合成酶识别的负向决定因素。在tRNAVal中插入G:U取代保守的U4:A69会降低氨酰化效率，这主要是由于Km增加。当G:U位于3:70位置时，氨酰化效率会有较小但显著的下降；在受体螺旋的其他位置观察到的G:U影响较小。通过对5 - 氟尿嘧啶取代的tRNAVal进行19F NMR光谱监测，发现G:U碱基对的负面影响与4:69位置附近螺旋结构的变化密切相关。这表明在受体茎中维持规则的A型RNA螺旋几何结构对于缬氨酰 - tRNA合成酶正确识别tRNAVal很重要。19F NMR还表明，tRNAVal - 缬氨酰 - tRNA合成酶复合物的形成不会破坏受体茎中的第一个碱基对，这一结果与报道的tRNAGln - 谷氨酰胺 - tRNA合成酶复合物不同。
