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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 氨基酰基-TRNA合成酶（AARSS）是一个由20个必需酶的家族，负责将20个氨基酸连接到其同源转移RNA（TRNA）中，以高度具体的反应，从而影响所有活细胞中遗传密码的翻译。乙酰基-TRNA Snythetase（Alars）将丙氨酸连接到两个步骤的tRNA（ALA）上。首先，将酶结合的丙氨酸被ATP激活，然后将丙基甲基甲基酸酯部分转移至酶结合的tRNA（ALA），释放AMP和焦磷酸盐。在AARS中，Alars在功能上是唯一的。例如，Alars在tRNA受体茎上不需要几分（七个）碱基对，以用丙氨酸特异性氨基酰胺化，并且对tRNA的反密码子序列无关紧要。 TRNAALA身份的主要确定源是位于受体螺旋中的单个摇摆基碱对G3：U70。这对基对的突变可预防体外和体内氨基酰化及其在非共同trNA中的包含，使它们能够接受丙氨酸。我们要揭示Alars这些独特功能特性的结构基础。在20个AARS中，Alars仍然是唯一没有已知晶体结构的合成酶。这很大，由于难以结晶酶。使用一种新型的高通量结晶方法，我们最近从极端热嗜热aquifex aeolicus获得了Alars的催化片段的天然和SEL-MET标记的晶体。晶体在内部设备上衍射至3.0»，在空间群P2（1）2（1）2中掉落，在非对称单元中有一个分子。通过将小的RN​​A底物扩散到自由酶的晶体中，制备了带有RNA的复杂晶体。我们的目标是使用MAD方法确定Alars催化片段的从头晶体结构和与RNA的复合物。为此，我们希望在SSRL收集必要的SE-MAD数据。