Larkin Deana C, Williams Amy M, Martinis Susan A, Fox George E
Department of Biology and Biochemistry, 369 Science and Research Building II, University of Houston, Houston, TX 77204-5001, USA.
Nucleic Acids Res. 2002 May 15;30(10):2103-13. doi: 10.1093/nar/30.10.2103.
Escherichia coli leucyl-tRNA synthetase (LeuRS) aminoacylates up to six different class II tRNA(leu) molecules. Each has a distinct anticodon and varied nucleotides in other regions of the tRNA. Attempts to construct a minihelix RNA that can be aminoacylated with leucine have been unsuccessful. Herein, we describe the smallest tRNA(leu) analog that has been aminoacylated to a significant extent to date. A series of tRNA(leu) analogs with various domains and combinations of domains deleted was constructed. The minimal RNA that was efficiently aminoacylated with LeuRS was one in which the anticodon stem-loop and variable arm stem-loop, but neither the D-arm nor T-arm, were deleted. Aminoacylation of this minimal RNA was abolished when the discriminator base A73 was replaced with C73 or when putative tertiary interactions between the D-loop and T-loop were disrupted, suggesting that these identity elements are still functioning in the minimized RNA. The various constructs that were significantly aminoacylated were also tested for amino acid editing by the synthetase. The anticodon and variable stem-loop domains were also dispensable for hydrolysis of the charged tRNA(leu) mimics. These results suggest that LeuRS may rely on identity elements in overlapping domains of the tRNA for both its aminoacylation and editing activities.
大肠杆菌亮氨酰 - tRNA合成酶（LeuRS）可将多达六种不同的II类tRNA(leu)分子氨酰化。每种tRNA(leu)都有独特的反密码子，且在tRNA的其他区域具有不同的核苷酸。构建能够被亮氨酸氨酰化的小螺旋RNA的尝试均未成功。在此，我们描述了迄今为止已被显著氨酰化的最小的tRNA(leu)类似物。构建了一系列缺失不同结构域及其组合的tRNA(leu)类似物。能够被LeuRS有效氨酰化的最小RNA是一种缺失了D臂和T臂，但保留了反密码子茎环和可变臂茎环的RNA。当鉴别碱基A73被替换为C73时，或者当D环和T环之间假定的三级相互作用被破坏时，这种最小RNA的氨酰化作用就会消失，这表明这些识别元件在最小化的RNA中仍在发挥作用。对各种被显著氨酰化的构建体也进行了合成酶的氨基酸编辑测试。反密码子和可变茎环结构域对于带电荷的tRNA(leu)模拟物的水解也是可有可无的。这些结果表明，LeuRS的氨酰化和编辑活性可能依赖于tRNA重叠结构域中的识别元件。
