Anticodon loop size and sequence requirements for recognition of formylmethionine tRNA by methionyl-tRNA synthetase.
Anticodon loop size and sequence requirements for recognition of formylmethionine tRNA by methionyl-tRNA synthetase.
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甲硫氨酰-tRNA 合成酶识别甲酰甲硫氨酸 tRNA 的反密码子环大小和序列要求。
DOI：
10.1073/pnas.80.22.6755
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发表时间：
1983
影响因子：
11.1
通讯作者：
Pelka,H
中科院分区：
文献类型：
--
作者：
Schulman,LH;Pelka,H
本实验室以前的工作确定了大肠杆菌tRNAfMet中几个对大肠杆菌识别该tRNA至关重要的特异性位点。coli甲硫氨酰-tRNA合成酶（EC 6.1.1.10）。特别强有力的证据表明，在反密码子的摆动位置的核苷酸碱基在歧视过程中的作用。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的每个位置含有单碱基变化的tRNAfMet衍生物。此外，已经制备了含有置换序列和更大和更小的反密码子环的衍生物。已经在体外酶促合成了变体tRNA。该过程涉及切除正常的反密码子，CAU，通过有限的消化完整的tRNAfMet与胰腺RNA酶。该步骤还从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，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的合成衍生物以与完整tRNAfMet相同的动力学被氨酰化。在摆动位置的碱基取代降低氨酰化速率至少五个数量级。在反密码子的其他两个位置具有碱基取代的衍生物的氨酰化速率是正常的1/55至1/18，500倍。具有与正常反密码子碱基相同的特定官能团的核苷酸在这些位置中的每一个上比不具有这些官能团的核苷酸更好地耐受。具有仅含有反密码子的CA序列的六元环的tRNAfMet变体的氨酰化更慢，并且含有五元环的衍生物没有可测量的活性。正常的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重要。我们从这些数据中得出结论，tRNAfMet的识别需要甲硫氨酰-tRNA合成酶与反密码子序列的核苷酸碱基上的官能团的高度特异性的相互作用。
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.
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DOI：
10.1073/pnas.79.4.1240
发表时间：
1982
影响因子：
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作者：
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DOI：
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发表时间：
1984
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
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作者：
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影响因子：
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