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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。氨基酰基trna合成酶（aaRSs）是一个由20种必需酶组成的家族，它们负责在高度特异性的反应中将20种氨基酸连接到其同源转移rna （tRNAs）上，从而影响所有活细胞中遗传密码的翻译。Alanyl-tRNA snythetase （AlaRS）通过两步将丙氨酸连接到tRNAAla上。首先，酶结合的丙氨酸被ATP激活，然后丙烯酰腺苷酸部分被转移到酶结合的tRNAAla上，释放AMP和焦磷酸。在aars中，AlaRS的功能是独一无二的。例如，AlaRS需要tRNA受体中不超过几个（7个）碱基对来与丙氨酸进行特异性的氨基酰化，并且对tRNA的反密码子序列无关。tRNAAla身份的主要决定因素是位于受体螺旋中的单个摆动碱基对G3:U70。该碱基对的突变在体外和体内阻止了氨基酰化，而它在非同源trna中的包含使它们能够接受丙氨酸。我们想要揭示AlaRS这些独特功能特性的结构基础。在20种aars中，AlaRS是唯一没有已知晶体结构的合成酶。这主要是由于酶结晶困难。利用一种新的高通量结晶方法，我们最近从极端嗜热性水蛭（Aquifex aeolicus）中获得了AlaRS催化片段的天然和自met标记晶体。晶体在内部设备上衍射到3.0°，落在P21212空间群中，其中一个分子在不对称单元中。通过将小RNA底物扩散到游离酶的存在晶体中制备RNA复合晶体。我们的目标是使用MAD方法确定AlaRS催化片段和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。

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 tRNAAla in two-steps. First, enzyme-bound alanine is activated by ATP, then the alanyl-adenylate moiety is transferred to enzyme-bound tRNAAla, 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 P21212, 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.