Mimics of yeast tRNAAsp and their recognition by aspartyl-tRNA synthetase.
Mimics of yeast tRNAAsp and their recognition by aspartyl-tRNA synthetase.
复制标题
酵母 tRNAAsp 的模拟物及其被天冬氨酰-tRNA 合成酶的识别。
DOI：
10.1021/bi9908383
复制
发表时间：
1999
期刊：
影响因子：
2.9
通讯作者：
R. Giegé
中科院分区：
文献类型：
--
作者：
A. Wolfson;A. Khvorova;C. Sauter;C. Florentz;R. Giegé
假设 tRNA 的 L 形三维结构是一个结构框架，允许将同一核苷酸正确呈递给氨酰基-tRNA 合成酶，这意味着改变和/或简化的 RNA 结构可以发挥这一作用，并成为这些酶的功能底物，前提是它们含有正确定位的同一元件。在这项工作中，该范式被提交给新的实验验证。酵母天冬氨酰-tRNA 合成酶是模型合成酶，研究了在不丧失其氨酰化能力的情况下可以改变同源 tRNAAsp 的规范结构框架的程度。发现了合成酶识别的三种新颖结构。第一个类似于缺乏 D 臂的后生动物线粒体 tRNASer。第二个缺乏 D 臂和 T 臂，以及氨基酸受体臂的 5' 链。第三种结构是受体螺旋和反密码子螺旋通过两个连接器连接的构建体。这些 RNA 的天冬氨酸化特异性通过假定的同一残基突变时氨酰化活性的丧失来验证。动力学数据表明，前两种结构是整个 tRNAAsp 分子的模拟物，而第三种结构则表现为天冬氨酸小螺旋模拟物。结果证实了鉴别核苷酸 G73 在天冬氨酸化中的原始作用，并证明受体结构域中的螺旋结构和 D 臂或 T 臂的存在都不是特定天冬氨酸化所必需的，但该活性依赖于反密码子环中同源天冬氨酸 GUC 序列的存在。
Assuming that the L-shaped three-dimensional structure of tRNA is an architectural framework allowing the proper presentation of identity nucleotides to aminoacyl-tRNA synthetases implies that altered and/or simplified RNA architectures can fulfill this role and be functional substrates of these enzymes, provided they contain correctly located identity elements. In this work, this paradigm was submitted to new experimental verification. Yeast aspartyl-tRNA synthetase was the model synthetase, and the extent to which the canonical structural framework of cognate tRNAAsp can be altered without losing its ability to be aminoacylated was investigated. Three novel architectures recognized by the synthetase were found. The first resembles that of metazoan mitochondrial tRNASer lacking the D-arm. The second lacks both the D- and T-arms, and the 5'-strand of the amino acid acceptor arm. The third structure is a construct in which the acceptor and anticodon helices are joined by two connectors. Aspartylation specificity of these RNAs is verified by the loss of aminoacylation activity upon mutation of the putative identity residues. Kinetic data indicate that the first two architectures are mimics of the whole tRNAAsp molecule, while the third one behaves as an aspartate minihelix mimic. Results confirm the primordial role of the discriminator nucleotide G73 in aspartylation and demonstrate that neither a helical structure in the acceptor domain nor the presence of a D- or T-arm is mandatory for specific aspartylation, but that activity relies on the presence of the cognate aspartate GUC sequence in the anticodon loop.