Fluorine-19 nuclear magnetic resonance as a probe of the solution structure of mutants of 5-fluorouracil-substituted Escherichia coli valine tRNA.
Fluorine-19 nuclear magnetic resonance as a probe of the solution structure of mutants of 5-fluorouracil-substituted Escherichia coli valine tRNA.
复制标题
氟 19 核磁共振作为 5-氟尿嘧啶取代大肠杆菌缬氨酸 tRNA 突变体溶液结构的探针。
DOI：
10.1016/0022-2836(92)90528-r
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发表时间：
1992
影响因子：
5.6
通讯作者：
Horowitz,J
中科院分区：
文献类型：
--
作者：
Chu,WC;Feiz,V;Derrick,WB;Horowitz,J
为了利用~(19)F核磁共振探测5-氟尿嘧啶标记的大肠杆菌tRNA-Val的溶液结构，我们对其~(19)F谱进行了归属。在这里，我们描述了通过检查一系列tRNA Val突变体的光谱来进行指定的，这些突变体具有单个5-氟尿嘧啶残基的核苷酸替换。并对碱基置换对tRNA结构和功能的影响进行了表征。对克隆的tRNAVal基因进行寡核苷酸定向突变，用噬菌体T7RNA聚合酶体外转录tRNAs。通过识别每个tRNA变体的19F核磁共振谱中缺失的峰，我们能够指定tRNA环区和茎区氟尿嘧啶残基的共振。由于FU33、FU34和FU29的归属，反密码子环和茎构象的变化可能导致光谱随温度的变化。对FU64共振的镁离子依赖性分裂的观察表明，tRNAVal的T臂可以在核磁共振时间尺度上以两种缓慢交换的构象存在。环和茎中大部分5-氟尿嘧啶残基的替换对tRNAVal的结构几乎没有影响；突变的tRNAs的19F核磁共振谱几乎没有移动。然而，用C29·G41替换反密码子茎中的FU29·A41碱基对会导致反密码子环和P-10环的构象变化。通过比较tRNAVal突变体和野生型tRNA的Vmax和Km值，确定核苷酸替代对氨基酰化的影响。反密码子3‘端(36位)的核苷酸替换使tRNA Val的氨基酰化效率(Vmax Km)降低了3个数量级。反密码子5‘端(34位)的碱基替换对氨酰化效率的影响很小。FU29·A41碱基对的取代使Km值增加了20倍，而Vmax几乎保持不变。受体茎中的FU4·A69碱基对很容易被替换，对大肠杆菌tRNA Val的氨酰化效率影响不大，表明该碱基对不是tRNA的同源元件。
In order to utilize 19 F nuclear magnetic resonance (NMR) to probe the solution structure of Escherichia coli tRNA Val labeled by incorporation of 5-fluorouracil, we have assigned its 19 F spectrum. We describe here assignments made by examining the spectra of a series of tRNA Val mutants with nucleotide substitutions for individual 5-fluorouracil residues. The result of base replacements on the structure and function of the tRNA are also characterized. Mutants were prepared by oligonucleotide-directed mutagenesis of a cloned tRNA Val gene, and the tRNAs transcribed in vitro by bacteriophage T7 RNA polymerase. By identifying the missing peak in the 19 F NMR spectrum of each tRNA variant we were able to assign resonances from fluorouracil residues in loop and stem regions of the tRNA. As a result of the assignment of FU33, FU34 and FU29, temperature-dependent spectral shifts could be attributed to changes in anticodon loop and stem conformation. Observation of a magnesium ion-dependent splitting of the resonance assigned to FU64 suggested that the T-arm of tRNA Val can exist in two conformations in slow exchange on the NMR time scale. Replacement of most 5-fluorouracil residues in loops and stems had little effect on the structure of tRNA Val; few shifts in the 19 F NMR spectrum of the mutant tRNAs were noted. However, replacing the FU29· A41 base-pair in the anticodon stem with C29· G41 induced conformational changes in the anticodon loop as well as in the P-10 loop. Effects of nucleotide substitution on aminoacylation were determined by comparing the V max and K m values of tRNA Val mutants with those of the wild-type tRNA. Nucleotide substitution at the 3′ end of the anticodon (position 36) reduced the aminoacylation efficiency (V max K m) of tRNA Val by three orders of magnitude. Base replacement at the 5′ end of the anticodon (position 34) had only a small negative effect on the aminoacylation efficiency. Substitution of the FU29· A41 base-pair increased the K m value 20-fold, while V max remained almost unchanged. The FU4· A69 base-pair in the acceptor stem, could readily be replaced with little effect on the aminoacylation efficiency of E. coli tRNA Val, indicating that this base-pair is not an identity element of the tRNA, as suggested by others.