DOMAINS OF HISTIDINE TRNA SYNTHETASE SUBSTRATE BINDING

描述：准确的蛋白质合成需要每个氨基酰基-tRNA 合成酶必须从化学上相似的底物中选择底物 分子。这种区别与催化之间的关系 函数是生物学中的一个重要问题。申请者的试验性 系统使用结构生物学、生物化学和分子遗传学来 研究大肠杆菌组氨酰-tRNA合成酶的结构和功能， 最小和最简单的II类氨酰-tRNA合成酶之一。vbl.使用 该体系、酶-底物和酶-产物复合体的结构 已经获得了。此外，组氨酰-tRNA合成酶的突变体 改变tRNA识别和其他催化显著差异 并定义了最小催化区域。 催化作用似乎是由两种“诱导配合”结合促进的， 适合后续反应的构象的底物，并通过使用 结合能来降低反应势垒。这些假设将是 通过研究结构-活性关系来进行测试 预稳态动力学在野生型和异构体反应中的应用 突变的酶。 所提出的研究目的是：1)确定序列特异性tRNA的基础 X射线结晶学和双突变热力学循环的识别 分析；2)探讨催化机理的本质特征 调查推测的催化残基，镁结合部位，以及 氨基酸选择的基础；以及3)表征结构和 具有催化活性的N-末端结构域相对于 二聚化、tRNA结合和特定的动力学缺陷。 在氨酰基-tRNA合成酶中，HisRS氨酰化产物种类繁多。 RNA底物，所以这些研究将对它们的贡献有价值 了解蛋白质对RNA的识别。此外，HisRS催化 结构域在结构上与翻译调节蛋白有关，并且 人类酶是一类自身免疫性疾病的主要抗原。 以针对蛋白质的自身抗体为特征的：RNA复合体。如果有资金支持 该项目将取代目前资助的R29，其目标是 大肠杆菌组氨酰-tRNA底物选择依据的研究 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.