Harris C L, Marashi F, Sakallah S
Department of Biochemistry, West Virginia University, School of Medicine, Morgantown 26506.
Biochim Biophys Acta. 1988 Jul 13;950(2):172-81. doi: 10.1016/0167-4781(88)90009-7.
Transfer RNA from Escherichia coli C6, a Met-, Cys-, relA- mutant, was previously shown to contain an altered tRNA(Ile) which accumulates during cysteine starvation (Harris, C.L., Lui, L., Sakallah, S. and DeVore, R. (1983) J. Biol. Chem. 258, 7676-7683). We now report the purification of this altered tRNA(Ile) and a comparison of its aminoacylation and chromatographic behavior and modified nucleoside content to that of tRNA(Ile) purified from cells of the same strain grown in the presence of cysteine. Sulfur-deficient tRNA(Ile) (from cysteine-starved cells) was found to have a 5-fold increased Vmax in aminoacylation compared to the normal isoacceptor. However, rates or extents of transfer of isoleucine from the [isoleucyl approximately AMP.Ile-tRNA synthetase] complex were identical with these two tRNAs. Nitrocellulose binding studies suggested that the sulfur-deficient tRNA(Ile) bound more efficiently to its synthetase compared to normal tRNA(Ile). Modified nucleoside analysis showed that these tRNAs contained identical amounts of all modified bases except for dihydrouridine and 4-thiouridine. Normal tRNA(Ile) contains 1 mol 4-thiouridine and dihydrouridine per mol tRNA, while cysteine-starved tRNA(Ile) contains 2 mol dihydrouridine per mol tRNA and is devoid of 4-thiouridine. Several lines of evidence are presented which show that 4-thiouridine can be removed or lost from normal tRNA(Ile) without a change in aminoacylation properties. Further, tRNA isolated from E. coli C6 grown with glutathione instead of cysteine has a normal content of 4-thiouridine, but its tRNA(Ile) has an increased rate of aminoacylation. We conclude that the low content of dihydrouridine in tRNA(Ile) from E. coli cells grown in cysteine-containing medium is most likely responsible for the slow aminoacylation kinetics observed with this tRNA. The possibility that specific dihydrouridine residues in this tRNA might be necessary in establishing the correct conformation of tRNA(Ile) for aminoacylation is discussed.
先前已证明，来自大肠杆菌C6（一种Met-、Cys-、relA-突变体）的转运RNA含有一种改变的tRNA（Ile），它在半胱氨酸饥饿期间积累（哈里斯，C.L.，刘，L.，萨卡拉，S.和德沃尔，R.（1983年）《生物化学杂志》258，7676 - 7683）。我们现在报告这种改变的tRNA（Ile）的纯化，并将其氨酰化、色谱行为和修饰核苷含量与从在半胱氨酸存在下生长的同一菌株细胞中纯化的tRNA（Ile）进行比较。发现硫缺乏的tRNA（Ile）（来自半胱氨酸饥饿细胞）在氨酰化中的Vmax比正常同功受体增加了5倍。然而，异亮氨酸从[异亮氨酰 - 大约AMP·异亮氨酸 - tRNA合成酶]复合物的转移速率或程度在这两种tRNA中是相同的。硝酸纤维素结合研究表明，与正常tRNA（Ile）相比，硫缺乏的tRNA（Ile）与其合成酶的结合更有效。修饰核苷分析表明，除了二氢尿苷和4 - 硫尿苷外，这些tRNA含有相同量的所有修饰碱基。正常tRNA（Ile）每摩尔tRNA含有1摩尔4 - 硫尿苷和二氢尿苷，而半胱氨酸饥饿的tRNA（Ile）每摩尔tRNA含有2摩尔二氢尿苷且不含4 - 硫尿苷。提供了几条证据表明，4 - 硫尿苷可以从正常tRNA（Ile）中去除或丢失而不改变氨酰化特性。此外，从用谷胱甘肽而非半胱氨酸培养的大肠杆菌C6中分离的tRNA具有正常含量的4 - 硫尿苷，但其tRNA（Ile）的氨酰化速率增加。我们得出结论，在含半胱氨酸培养基中生长的大肠杆菌细胞的tRNA（Ile）中二氢尿苷含量低最有可能是观察到该tRNA氨酰化动力学缓慢的原因。讨论了这种tRNA中特定的二氢尿苷残基对于建立用于氨酰化的tRNA（Ile）正确构象可能是必需的可能性。
