CRYSTAL STRUCTURE DETERMINATION OF THE ALANYL-TRNA SYNTHETASE AND ITS COMPLEXES

丙氨酰-TRNA合成酶及其复合物的晶体结构测定

• 批准号: 7721733
• 负责人: PAUL R SCHIMMEL
• 金额: $ 0.02万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721733
• 关键词: Affect; Alanine; Alanine-Specific tRNA; Amino Acids; Amino Acyl-tRNA Synthetases; Aminoacylation; Anticodon; Base Pairing; Binding; Cells; Complex; Computer Retrieval of Information on Scientific Projects Database; Crystallization; Data; Diffuse; Diphosphates; Enzymes; Equipment; Family; Funding; Genetic Code; Goals; Grant; Helix (Snails); Housing; In Vitro; Institution; Label; Life; Ligase; Methods; Mutation; Property; RNA; Reaction; Research; Research Personnel; Resources; Small RNA; Source; Structure; Transfer RNA; Translations; United States National Institutes of Health; adenylate; base; ear helix; extreme thermophile; falls; in vivo; novel; prevent; stem; tRNA-alanine complex

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Aminoacyl-tRNA synthetases (aaRSs) are a family of 20 essential enzymes responsible for attaching the 20 amino acids to their cognate transfer-RNAs (tRNAs) in a highly specific reaction, thereby affecting the translation of the genetic code in all living cells. Alanyl-tRNA snythetase (AlaRS) attaches alanine onto tRNA(Ala) in two-steps. First, enzyme-bound alanine is activated by ATP, then the alanyl-adenylate moiety is transferred to enzyme-bound tRNA(Ala), releasing AMP and pyrophosphate. Among aaRSs, AlaRS is functionally unique. For example, AlaRS requires no more than a few (seven) base pairs in the tRNA acceptor stem for specific aminoacylation with alanine and is indifferent toward the tRNA's anticodon sequence. The major determinant of the identity of tRNAAla is a single wobble base pair, G3:U70, located in the acceptor helix. Mutations in this base pair prevent aminoacylation in vitro and in vivo and its inclusion in non-cognate tRNAs enables them to accept alanine. We want to reveal the structural basis of these unique functional properties of AlaRS. Of the 20 aaRSs, AlaRS remains to be the only synthetase without a known crystal structure. This is largely due to difficulty in crystallizing the enzyme. Using a novel high-throughput crystallization approach, we recently obtained native and sel-Met-labelled crystals of a catalytic fragment of AlaRS from the extreme thermophile Aquifex aeolicus. The crystals diffract to 3.0 ¿ on in-house equipment and fall in the space group P2(1)2(1)2, with one molecule in the asymmetric unit. Complex crystals with RNA are prepared by diffusing small RNA substrates into exiting crystals of the free enzyme. Our goal is to determine the de novo crystal structures of the AlaRS catalytic fragment and complexes with RNA using MAD methods. To that end, we hope to collect the necessary Se-MAD data at SSRL.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 氨酰-tRNA 合成酶 (aaRS) 是一个由 20 种必需酶组成的家族，负责以高度特异性的反应将 20 种氨基酸附着到其同源转移 RNA (tRNA) 上，从而影响所有活细胞中遗传密码的翻译。 -tRNA 合成酶 (AlaRS) 将丙氨酸分两步附着到 tRNA(Ala) 上，首先，酶结合的丙氨酸被激活。 ATP，然后丙氨酰腺苷酸部分被转移到酶结合的 tRNA (Ala)，释放 AMP 和焦磷酸盐。 在 aaRS 中，AlaRS 在功能上是独特的，例如，AlaRS 在 tRNA 中只需要几个（七个）碱基对。受体茎与丙氨酸进行特异性氨酰化，并且对 tRNA 的反密码子序列无关 tRNAAla 身份的主要决定因素是单个摆动。碱基对 G3:U70 位于受体螺旋中，该碱基对的突变可防止体外和体内的氨酰化，并且其包含在非同源 tRNA 中，使它们能够接受丙氨酸。在 20 种 aaRS 中，AlaRS 仍然是唯一一种没有已知晶体结构的合成酶，这主要是由于使用新型高通量酶的结晶困难。通过结晶方法，我们最近从极端嗜热菌 Aquifex aeolicus 中获得了 AlaRS 催化片段的天然和 sel-Met 标记的晶体。晶体衍射至 3.0 ¿在内部设备上，属于空间群 P2(1)2(1)2，不对称单元中有一个分子，通过将小 RNA 底物扩散到游离酶的现有晶体中来制备。的目的是使用 MAD 方法确定 AlaRS 催化片段和与 RNA 复合物的从头晶体结构，为此，我们希望在 SSRL 收集必要的 Se-MAD 数据。