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。目标是解释中的功能 由高分辨率晶体衍生的立体化学细节术语 相关大分子及其络合物的结构。 1.真核生物蛋白质合成的启动：这里的目的是 解释真核生物启动子tRNA的特殊功能 酵母tRNA-i-Met的分子模型立体化学分析将会是 在不同的氨基酰化启动子tRNA上进行，以了解 氨基酸侧链的作用出乎意料的重要。站点定向 将使用突变技术(与其他人合作)来确定 序列要素负责的每个特殊属性 真核表达的Trn-Ai-Met。 2.tRNA/氨酰-tRNA合成酶：试图准备X射线质量 对晶体的络合物，将进行计算机图形学实验 将tRNA-i-Met的结构与Met-tRNA合成酶模型‘对接’。 主要的指南将是直接光交联剂和标记反应 它模拟了tRNA的U8与 合成酶。这些“系链”表示tRNA和 合成酶中相互接触的氨基酸。 络合物的分子界面。 3.细菌抑制：色氨酸抑制物/操纵子系统：最新 已确定的色氨酸抑制物的原子模型将被提炼和比较 对于非活性抑制物的高分辨率结构(无约束 色氨酸)和不活跃的假抑制子(用一种 竞争对手)，目前正在确定 结晶学上的。将继续努力改善水晶 抑制子和算子的复合体。X射线衍射分析将 当合适的晶体准备好后，继续进行。这些晶体结构如 以及来自其他实验室的突变分析应该有助于开发出 序列特异性DNA调控结合的分子机制 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)结合和修饰红霉素抗性蛋白 核糖体RNA中的一个特定残基，(2)单体RNA T7噬菌体家族的聚合酶/启动子系统和(3) 哺乳动物细胞系中的类固醇受体/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.