Schulman L H, Pelka H
Proc Natl Acad Sci U S A. 1983 Nov;80(22):6755-9. doi: 10.1073/pnas.80.22.6755.
Previous work from our laboratory identified several specific sites in Escherichia coli tRNAfMet that are essential for recognition of this tRNA by E. coli methionyl-tRNA synthetase (EC 6.1.1.10). Particularly strong evidence indicated a role for the nucleotide base at the wobble position of the anticodon in the discrimination process. To further investigate the structural requirements for recognition in this region, we have synthesized a series of tRNAfMet derivatives containing single base changes in each position of the anticodon. In addition, derivatives containing permuted sequences and larger and smaller anticodon loops have been prepared. The variant tRNAs have been enzymatically synthesized in vitro. The procedure involves excision of the normal anticodon, CAU, by limited digestion of intact tRNAfMet with pancreatic RNase. This step also removes two nucleotides from the 3' CpCpA end. T4 RNA ligase is used to join oligonucleotides of defined length and sequence to the 5' half-molecule and subsequently to link the 3' and modified 5' fragment to regenerate the anticodon loop. The final step of the synthesis involves repair of the 3' terminus with tRNA nucleotidyltransferase. The synthetic derivative containing the anticodon CAU is aminoacylated with the same kinetics as intact tRNAfMet. Base substitutions in the wobble position reduce aminoacylation rates by at least five orders of magnitude. The rates of aminoacylation of derivatives having base substitutions in the other two positions of the anticodon are 1/55 to 1/18,500 times normal. Nucleotides that have specific functional groups in common with the normal anticodon bases are better tolerated at each of these positions than those that do not. A tRNAfMet variant having a six-membered loop containing only the CA sequence of the anticodon is aminoacylated still more slowly, and a derivative containing a five-membered loop is not measurably active. The normal loop size can be increased by one nucleotide with a relatively small effect on the rate of aminoacylation, indicating that the spatial arrangement of the nucleotides is less critical than their chemical nature. We conclude from these data that recognition of tRNAfMet requires highly specific interactions of methionyl-tRNA synthetase with functional groups on the nucleotide bases of the anticodon sequence.
我们实验室之前的工作确定了大肠杆菌甲硫氨酰 - tRNA合成酶（EC 6.1.1.10）识别该tRNA所必需的大肠杆菌tRNAfMet中的几个特定位点。特别有力的证据表明，反密码子摆动位置的核苷酸碱基在识别过程中起作用。为了进一步研究该区域识别的结构要求，我们合成了一系列在反密码子每个位置含有单个碱基变化的tRNAfMet衍生物。此外，还制备了含有置换序列以及更大和更小反密码子环的衍生物。这些变体tRNA是在体外酶促合成的。该过程包括用胰核糖核酸酶有限消化完整的tRNAfMet，切除正常的反密码子CAU。这一步还从3' CpCpA末端去除两个核苷酸。T4 RNA连接酶用于将确定长度和序列的寡核苷酸连接到5'半分子上，随后连接3'和修饰的5'片段以再生反密码子环。合成的最后一步涉及用tRNA核苷酸转移酶修复3'末端。含有反密码子CAU的合成衍生物与完整的tRNAfMet具有相同的氨基酰化动力学。摆动位置的碱基替换使氨基酰化速率降低至少五个数量级。在反密码子其他两个位置有碱基替换的衍生物的氨基酰化速率是正常速率的1/55至1/18500倍。与正常反密码子碱基具有特定官能团相同的核苷酸在这些位置中的每一个位置比那些没有的核苷酸更能被耐受。具有仅包含反密码子CA序列的六元环的tRNAfMet变体氨基酰化仍然更慢，并且含有五元环的衍生物没有可测量的活性。正常环大小可以增加一个核苷酸，对氨基酰化速率的影响相对较小，这表明核苷酸的空间排列不如其化学性质关键。我们从这些数据得出结论，tRNAfMet的识别需要甲硫氨酰 - tRNA合成酶与反密码子序列核苷酸碱基上的官能团进行高度特异性的相互作用。
