GENETICALLY AND CHEMICALLY DERIVED MISSENSE SUPPRESSOR TRANSFER RNAS WITH ALTERED ENZYMIC AMINOACYLATION RATES
GENETICALLY AND CHEMICALLY DERIVED MISSENSE SUPPRESSOR TRANSFER RNAS WITH ALTERED ENZYMIC AMINOACYLATION RATES
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DOI：
10.1016/0022-2836(68)90406-3
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发表时间：
1968-01-01
影响因子：
5.6
通讯作者：
CURRY, JB
中科院分区：
文献类型：
--
作者：
CARBON, J;CURRY, JB
用亚硝酸对tRNAGGY进行部分脱氨，获得了一种在体外氨基酸掺入系统中特异性改变的转移RNA。这种改变的tRNA将[14 C]甘氨酸插入到多肽中，以响应交替的多核糖核苷酸poly（A-G），这种活性以前被证明是携带色氨酸合成酶A蛋白中突变A36抑制因子的大肠杆菌菌株的tRNA的特征。基因（su 36+）和化学衍生的抑制tRNA都将精氨酸密码子阿加读作甘氨酸密码子。化学衍生的“抑制剂”的制备方法的改进已经确定活性来自tRNAGGGly的变化。反密码子的改变（UCC→UCU）可能是相关的，但这方面的直接证据仍然缺乏。对遗传和化学衍生的抑制子的色谱比较表明它们是不同的分子种类，尽管在两个系统中都在tRNAGly区色谱。以前的迹象表明，su 36+抑制剂在甘氨酸活化酶催化的氨酰化反应中反应缓慢，已在体外得到证实。两种抑制剂的酶促充电的实际速率已经通过利用高碘酸盐氧化来测量，以在由各种水平的纯化甘氨酰tRNA合成酶催化的加载反应中，在给定时间后使未氨酰化的部分脱氨。在37 °C下，30分钟内将相同数量的正常tRNAGly氨酰化，需要大约4 × 105倍的酶来完成这些抑制剂的负载。当校正浓度效应时，似乎在酶促氨酰化反应中（在饱和水平的tRNA下），su 36+和化学衍生的抑制性tRNA的反应比正常的tRNAGly慢约2 × 103倍。
A transfer RNA species with altered specificity in anin vitroamino acid-incorporating system has been obtained by partial deamination of tRNAGGAGlywith nitrous acid. This altered tRNA inserts [14C]glycine into polypeptides in response to the alternating polyribonucleotide, poly (A-G), an activity previously shown to be characteristic of tRNA fromEscherichia colistrains carrying a suppressor of mutationA36in the tryptophan synthetase A protein. Both the genetically (su36+) and chemically derived suppressor tRNA's read the arginine codon, AGA, as a glycine codon. Improvements in the method of preparation of the chemically derived “suppressor” have established that the activity arises from a change in a tRNAGGAGly. An anticodon change (UCC→UCU) is probably involved, but direct evidence of this is still lacking.Chromatographic comparisons of the genetically and chemically derived suppressors have shown them to be different molecular species, although both chromatograph in the tRNAGlyregion in two systems. Previous indications that thesu36+suppressor reacts slowly in the aminoacylation reaction catalyzed by the glycine activating enzyme have been confirmedin vitro. The actual rates of enzymic charging of the two suppressors have been measured by making use of periodate oxidation to inactivate that fraction not aminoacylated after a given time in loading reactions catalyzed by various levels of purified glycyl tRNA synthetase. It requires approximately 4 × 105times more enzyme to load these suppressors to completion than is required to aminoacylate the same quantity of normal tRNAGlyin 30 minutes at 37 °C. When corrected for concentration effects, it appears that both thesu36+and chemically derived suppressor tRNA's react approximately 2 × 103times slower than does normal tRNAGlyin the enzymic aminoacylation reaction (at saturating levels of tRNA).