Schulman L H, Pelka H
Fed Proc. 1984 Dec;43(15):2977-80.
In previous work we identified several specific sites in Escherichia coli tRNAfMet that are essential for recognition of this tRNA by E. coli methionyl-tRNA synthetase (MetRS) (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. We have now investigated the aminoacylation activity of 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 by using T4 RNA ligase (EC 6.5.1.3). Base substitutions in the wobble position have been found to reduce aminoacylation rates by at least five orders of magnitude. Derivatives having base substitutions in the other two positions of the anticodon are aminoacylated 55-18,500 times slower than 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, which indicates 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 MetRS with functional groups on the nucleotide bases of the anticodon sequence. Several other aminoacyl-tRNA synthetases are known to require one or more anticodon bases for efficient aminoacylation of their tRNA substrates, and data from other laboratories suggest that anticodon sequences may be important for accurate discrimination between cognate and noncoagnate tRNAs by these enzymes.
在之前的工作中，我们确定了大肠杆菌甲硫氨酰 - tRNA合成酶（MetRS）（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。我们现在研究了一系列在反密码子每个位置含有单碱基变化的tRNAfMet衍生物的氨酰化活性。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。通过使用T4 RNA连接酶（EC 6.5.1.3）在体外酶促合成了变体tRNA。已发现摆动位置的碱基替换可使氨酰化速率降低至少五个数量级。在反密码子其他两个位置具有碱基替换的衍生物的氨酰化速度比正常情况慢55 - 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都比不具有这些官能团的核苷酸更能被容忍。一种具有仅包含反密码子CA序列的六元环的tRNAfMet变体的氨酰化速度更慢，而一种含有五元环的衍生物则没有可测量的活性。正常环大小可以增加一个核苷酸，对氨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，MetRS对tRNAfMet的识别需要MetRS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。已知其他几种氨酰 - tRNA合成酶在其tRNA底物的有效氨酰化过程中需要一个或多个反密码子碱基，并且其他实验室的数据表明，反密码子序列对于这些酶准确区分同源和非同源tRNA可能很重要。
