STRUCTURAL ANALYSIS OF BIOLOGICAL MACROMOLECULES

我们将继续将结构/功能研究应用于以下三个方面： 基因表达和调控。 目的是解释函数在 从高分辨晶体导出的立体化学细节术语 相关大分子及其复合物的结构。 1.真核生物中蛋白质合成的起始：这里的目的是 解释真核生物起始tRNA的特殊功能， 酵母tRNA-i-Met的分子模型。 立体化学分析将是 对各种错氨酰化的起始物tRNA进行了研究，以了解 氨基酸侧链的重要作用。 定点 将采用诱变（与其他人合作）来确定 序列特征负责每个特殊属性， 真核生物tRN-Ai-Met。 2. tRNA/氨酰-tRNA合成酶：在试图制备X射线质量的同时 晶体的复杂性，计算机图形实验将进行 将tRNA-i-Met的结构与Met-tRNA合成酶的模型“对接”。 主要的指导将是直接的光交联和标记反应 它模拟了tRNA的U8与 合成酶 这些“系链”表示tRNA的核苷酸， 合成酶的氨基酸，其在所述酶的下游彼此接触， 复合物的分子界面。 3.细菌阻遏：trp阻遏子/操纵子系统：最近 确定的色氨酸阻遏物的原子模型将被细化和比较 与失活的去辅阻遏物的高分辨率结构（没有结合 色氨酸）和无活性的假阻遏物（色氨酸被色氨酸取代）。 竞争对手），目前正在确定 晶体学上。 将继续努力提高结晶 阻遏物和操纵子的复合物。 X射线衍射分析将 准备好合适的晶体后继续进行。 这些晶体结构， 以及其他实验室的突变分析应该有助于开发一种 调控序列特异性DNA结合的分子机制 蛋白质和小配体对遗传调节蛋白的调节。 将审查未来结构研究的新监管系统， 包括：（1）红霉素抗性蛋白， 核糖体RNA中的特定残基，（2）单体RNA 来自噬菌体T7家族的聚合酶/启动子系统，和（3） 类固醇受体/增强子系统。

We will continue to apply structure/function studies to three aspects of genetic expression and regulation. The goal is to explain function in terms of stereochemical details derived from high resolution crystal structures of relevant macromolecules and their complexes. 1. Initiation of protein synthesis in eukaryotes: Here the aim is to explain the special functions of eukaryotic initiator tRNA in terms of the molecular model of yeast tRNA-i-Met. A stereochemical analysis will be carried out on various misaminoacylated initiator tRNAs to understand the unexpectedly important role of the amino acid side chain. Site-directed mutagenesis will be employed (in collaboration with others) to define which sequence features are responsible for each of the special attributes of eukaryotic tRN-Ai-Met. 2. tRNA/aminoacyl-tRNA synthetase: While trying to prepare X-ray quality crystals of the complex, computer graphic experiments will be carried out to 'dock' the structure of tRNA-i-Met with a model of Met-tRNA synthetase. The principal guides will be direct photocrosslinks and a labeling reaction that simulates the transient reaction of the tRNA's U8 with the synthetase. These "tethers" indicate the nucleotides of the tRNA and the amino acids of the synthetase that are in contact with one another at the molecular interface of the complex. 3. Bacterial repression: trp repressor/operator system: The recently determined atomic model of the trp repressor will be refined and compared to the high resolution structures of the inactive aporepressor (no bound tryptophan) and the inactive pseudorepressor (trytophan replaced by a competing antagonist) that are currently being determined crystallographically. Efforts will continue to improve the crystalline complexes of repressor and operator. X-ray diffraction analysis will proceed when suitable crystals are prepared. These crystal structures as well as mutational analyses from other labs should help to develop a molecular mechanism for sequence-specific DNA-binding by regulatory proteins and the modulation of genetic regulatory proteins by small ligands. New regulatory systems for future structural studies will be examined, including; (1) The erythromycin resistance protein that binds and modifies a specific residue in ribosomal RNA, (2) the monomeric RNA polymerase/promoter system from the T7 family of bacteriophage and (3) steroid receptor/enhancer system from mammalian cell lines.