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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。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（tRNA）上，从而影响所有活细胞中遗传密码的翻译。丙氨酰-tRNA合成酶（AlaRS）分两步将丙氨酸连接到tRNAAla上。首先，酶结合的丙氨酸被ATP激活，然后丙氨酰-腺苷酸部分转移到酶结合的tRNAAla，释放AMP和焦磷酸。在aaRS中，AlaRS在功能上是独特的。例如，AlaRS需要不超过几个（七个）碱基对的tRNA受体茎特异性氨酰化与丙氨酸，并对tRNA的反密码子序列无关。确定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：U 70。该碱基对中的突变阻止体外和体内的氨酰化，并且其包含在非同源tRNA中使其能够接受丙氨酸。我们希望揭示AlaRS这些独特功能特性的结构基础。在20种aaRS中，AlaRS仍然是唯一没有已知晶体结构的合成酶。这在很大程度上是由于酶结晶的困难。使用一种新的高通量结晶方法，我们最近获得了本地和sel-Met标记的晶体的催化片段AlaRS极端嗜热菌Aquifex aeolicus。该晶体在内部设备上的反应速率为3.0，属于空间群P21212，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现有晶体中来制备具有RNA的复合物晶体。我们的目标是确定从头晶体结构的AlaRS催化片段和复合物与RNA使用MAD方法。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.