TRP TRNA LIGASE--X-RAY STUDIES OF THE CATALYTIC CYCLE

我们的目标是关联的构象变化，在氨酰化的 tRNA具有特异性识别和底物转化作用。 我们 我希望测试构象变化的具体预测， 观察到嗜热脂肪B杆菌双氢酰-tRNA合成酶（TrpRS） 从无配体酶到Trp-5' AMP复合物位置 tRNA反密码子结合位点适合于酰基转移，相对于 另一个单体的活性位点。 为此，我们将解决新的 TrpRS与同源tRNA和ATP复合物的X射线结构。 在上一个资助周期中，我们解决了无配体酶， 色氨酸复合物;一种激活的基态三元复合物 与ATP和物种特异性抑制剂吲哚霉素;天然的 腺苷酸中间体Trp-5 'AMP和产物Dahanyl-2' 3 '-ATP。 我们将延长Trp-5' AMP的分辨率和实验阶段 复杂到其衍射极限，其优于1.7埃单位， 精确地描述了N- 单体的罗斯曼折叠结构域的末端螺旋的两个结构域， 它显然将活性位点行为与远端反密码子偶联 通过Ile结合位点16。Ile 16将突变为缬氨酸、亮氨酸， 丙氨酸来检验这个残基与小分子 含有反密码子结合位点的结构域。 我们将完成 从上面的列表中细化每个相关结构， 以产生用于氨基酸活化的结构反应曲线， tRNA的酰化。 特别感兴趣的是二聚体的变化， 通过将tRNA的受体茎结合到一个 活性位点和反密码子连接到另一个单体上的位点。 到 解释反密码子突变体如何抑制色氨酸 的trRNA杜松子酒，我们将比较复杂的与以前获得的 对于GlnRS。 原核生物TrpRS是抗感染药物的潜在靶点 药物发现，由于原核生物特异性 抑制剂吲哚霉素。 我们将研究高- 通过比较几种这样的复合物的亲和结合， 通过将我们的结构分析扩展到古细菌， 和真核TrpRS。

Our goal is to correlate conformation changes during aminoacylation of tRNA by with specific recognition and substrate transformations. We wish to test the specific prediction that conformational changes observed for B stearothermophilus tryptophanyl-tRNA synthetase (TrpRS) on proceeding from ligand-free enzyme to the Trp-5' AMP complex position the tRNA anticodon-binding site suitably for acyl-transfer, relative to the active site of the other monomer. To this end, we will solve new X-ray structures of TrpRS complexes with the cognate tRNA and with ATP. During the previous funding cycle we solved the ligand-free enzyme and complexes with tryptophan; an activated ground-state ternary complex with ATP and the species-specific inhibitor, indolmycin; the natural adenylate intermediate, Trp-5'AMP, and a product, tryptophanyl-2'3'-ATP. We will extend the resolution and experimental phases for the Trp-5' AMP complex to its diffraction limit which better than 1.7 Angstrom units, to precisely specific sidechain packing interactions between the N- terminal helix of the Rossmann-fold domain two domains of the monomer, which apparently couple active-site behavior to the distal anticodon binding site via Ile 16. Ile 16 will be mutated to valine, leucine, and alanine to test that hypothesis that this residue couples the small domain containing the anticodon-binding site to. We will finish refining each of the relevant structures from the above list, in order to produce a structural reaction profile for aminoacid activation and acylation of tRNA. Of special interest will be changes at the dimer interface introduced by binding the acceptor stem of the tRNA to one active site and the anticodon to a site on the other monomer. To explain how suppression with tryptophan occurs with an anticodon mutant of trRNA gin, we will compare the complex with the previously obtained for GlnRS. Prokaryote TrpRS is a potentially valuable target for anti-infective drug discovery, owing to the availability of a prokaryote-specific inhibitor, indolmycin. We will examine the structural bases for high- affinity binding by comparison of several such complexes, and the bases for specificity by extending our structural analysis to archebacterial and eukaryotic TrpRSs.