DOMAINS OF HISTIDINE TRNA SYNTHETASE SUBSTRATE BINDING

准确的蛋白质合成需要每个氨酰-tRNA 合成酶必须从化学性质相似的 分子。 这种区分和催化剂之间的关系 功能是生物学中的重要问题。 申请人的实验 系统使用结构生物学、生物化学和分子遗传学， 研究E.大肠杆菌组氨酰-tRNA合成酶， 最小和最简单的II类氨酰-tRNA合成酶之一。 使用 该系统，酶-底物和酶-产物复合物的结构 已经获得。 此外，具有以下特征的组氨酰-tRNA合成酶的突变体： 改变的tRNA识别和其他催化显著差异 的特征和最小催化域已被定义。 催化作用似乎是由两个“诱导适合”的结合， 适合于后续反应的构象的底物，以及通过使用 结合能降低反应势垒。 这些假设将是 通过调查结构-活性关系进行测试 预稳态动力学在野生型和野生型反应中的应用 突变酶 这些研究的目的是：1）确定序列特异性tRNA的基础 X射线晶体学和双突变热力学循环识别 分析; 2）探索催化机理的基本特征， 调查推定的催化残留物，镁结合位点，和 氨基酸选择的基础;和3）表征结构， 催化活性N-末端结构域的功能 二聚化、tRNA结合和特异性动力学缺陷。 在氨酰-tRNA合成酶中，HisRS使多种氨酰化。 RNA底物，因此这些研究将是有价值的，因为它们有助于 理解蛋白质对RNA的识别。 此外，HisRS催化 结构域在结构上与翻译调节蛋白相关， 人类酶是一类自身免疫性疾病的主要抗原 其特征在于针对蛋白质：RNA复合物的自身抗体。 如果得到资助 该项目将取代目前资助的R29，其目标是 探讨了E.大肠杆菌组氨酰-tRNA 合成酶

DESCRIPTION: Accurate protein synthesis requires that each aminoacyl-TRNA synthetase must select its substrates from a pool of chemically similar molecules. The relationship between this discrimination and catalytic function is an important problem in biology. The applicant's experimental system uses structural biology, biochemistry, and molecular genetics to investigate the structure and function of E. coli histidyl-TRNA synthetase, one of the smallest and simplest class II aminoacyl-TRNA synthetases. Using this system, structures of enzyme-substrate and enzyme-product complexes have been obtained. In addition, mutants of histidyl-TRNA synthetase with altered TRNA recognition and other catalytically significant differences have been characterized and the minimal catalytic domain has been defined. Catalysis appears to be promoted by both "induced fit" binding which places substrates in conformations suitable for subsequent reaction, and by the use of binding energy to lower reaction barriers. These hypotheses will be tested by investigating structure-activity relationships through the application of pre-steady state kinetics to the reactions of wild-type and mutant enzymes. The proposed studies aim to: 1) define the basis of sequence specific TRNA recognition by X-ray crystallography and double mutant thermodynamic cycle analysis; 2) probe the essential features of the catalytic mechanism by investigating putative catalytic residues, the magnesium binding site, and the basis of amino acid selection; and 3) characterize the structure and function of the catalytically active N-terminal domain with respect to dimerization, TRNA binding, and specific kinetic defects. Among the aminoacyl-TRNA synthetases, HisRS aminoacylates a wide variety of RNA substrates, so these studies will be valuable for their contribution to understanding RNA recognition by proteins. Moreover, the HisRS catalytic domain is structurally related to translational regulatory proteins, and the human enzyme is a major antigen in a class of autoimmune diseases characterized by autoantibodies against protein:RNA complexes. If funded this project would replace the currently funded R29, whose aims have been to investigate the basis of substrate selection by E. coli histidyl-TRNA synthetase.