亚硫酸氢盐诱导的C到U转换对大肠杆菌甘氨酸tRNA氨酰化的影响。

The effect of bisulfite-induced C to U transitions on aminoacylation of Escherichia coli glycine tRNA.

作者信息

Sabban E L, Bhanot O S

出版信息

J Biol Chem. 1982 May 10;257(9):4796-805.

Abstract

The effect of bisulfite-induced C to U transitions on the aminoacylation of highly purified Escherichia coli tRNAgly3,gcc has been studied. On treatment with 3.2 M NaHSO3 at 25 degrees C and pH 5.8, C to U transitions occurred at 4 of the 21 cytosine residues during the first 16 h. After about 12 h, 2 additional unidentified residues begin to react. From these data and theoretical consideration, we conclude: 1) E. coli tRNAgly3,gcc exists in the native conformation under the conditions of the reaction; 2) its ordered structure is similar to yeast tRNAPhe; 3) its anticodon loop is flexible in solution. During the reaction, loss of glycine acceptor activity followed first order kinetics with a t1/2 = 1.8 h. After modification for 1.25 h and aminoacylation (61% glycine acceptor activity remaining), the aminoacylated fraction was isolated. The fractional change at each of the reactive residues in the unfractionated mixture (f) and the aminoacylated fraction (p) was measured. The results were: C35, f = 0.33, p = 0.19; C36, f = 0.25, p = 0.10; C74, f = 0.28, p = 0.26; C75, f = 0.25, p = 0.20. From these data, the Modulation Constant for each reactive residue was calculated from the equation M = (f - p)/f(1 - p): M35 (anticodon) = 0.52; M36 (anticodon) = 0.67; M74 (CCA end) = 0.10; M75 (CCA end) = 0.25. These values, which are based on the assumptions that the C to U reactions occur independently and that the effect of each change on the acceptor activity is an independent event, express the fractional loss in activity that would occur from a C to U change at the residue in question by itself. From these results, we conclude: 1) approximately 80% of the observed inactivation was due to changes in the anticodon; 2) neither of these anticodon residues (C35 and C36) is essential for aminoacylation; 3) a C to U change at C75 (CCA end) has a small effect on aminoacylation; 4) a C to U change at C74 (CCA end) has little or no effect on aminoacylation.

摘要

研究了亚硫酸氢盐诱导的C到U转变对高度纯化的大肠杆菌tRNAgly3,gcc氨酰化的影响。在25℃和pH 5.8条件下用3.2 M NaHSO3处理时，21个胞嘧啶残基中的4个在最初16小时内发生了C到U的转变。大约12小时后，另外2个未鉴定的残基开始反应。根据这些数据和理论考虑，我们得出以下结论：1）大肠杆菌tRNAgly3,gcc在反应条件下以天然构象存在；2）其有序结构类似于酵母tRNAPhe；3）其反密码子环在溶液中是灵活的。在反应过程中，甘氨酸接受活性的丧失遵循一级动力学，t1/2 = 1.8小时。修饰1.25小时并进行氨酰化（剩余61%的甘氨酸接受活性）后，分离出氨酰化部分。测量了未分级混合物（f）和氨酰化部分（p）中每个反应性残基的分数变化。结果如下：C35，f = 0.33，p = 0.19；C36，f = 0.25，p = 0.10；C74，f = 0.28，p = 0.26；C75，f = 0.25，p = 0.20。根据这些数据，从方程M = (f - p)/f(1 - p)计算每个反应性残基的调节常数：M35（反密码子）= 0.52；M36（反密码子）= 0.67；M74（CCA末端）= 0.10；M75（CCA末端）= 0.25。这些值基于C到U反应独立发生以及每个变化对接受活性的影响是独立事件的假设，表达了在所讨论的残基处由C到U变化单独引起的活性分数损失。根据这些结果，我们得出以下结论：1）观察到的失活中约80%是由于反密码子的变化；2）这两个反密码子残基（C35和C36）对于氨酰化都不是必需的；3）C75（CCA末端）处的C到U变化对氨酰化有较小影响；4）C74（CCA末端）处的C到U变化对氨酰化几乎没有影响。