STRUCTURAL ANALYSIS OF BIOLOGICAL MACROMOLECULES

生物大分子结构分析

• 批准号: 3268760
• 负责人: PAUL B SIGLER
• 金额: $ 35.51万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-01-01 至 1991-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3268760
• 关键词: DNA binding protein; Escherichia coli; X ray crystallography; affinity chromatography; aminoacid tRNA ligase; bacterial RNA; bacterial genetics; bacterial proteins; binding proteins; computer graphics /printing; crosslink; enzyme substrate complex; erythromycin; gene expression; genetic operator element; genetic regulation; genetic transcription; nuclear magnetic resonance spectroscopy; nucleic acid sequence; nucleic acid structure; protein biosynthesis; radiotracer; ribosomes; site directed mutagenesis; transfer RNA; tryptophan

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.

我们将继续将结构/功能研究应用于 遗传表达和调节。 目的是解释功能 从高分辨率晶体得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启动：这里的目的是 用 酵母TRNA-I-MET的分子模型。 立体化学分析将是 在各种错误的启动器trnas上进行 氨基酸侧链的意外重要作用。 站点定向 将使用诱变（与他人合作）来定义哪个 序列特征是负责的每个特殊属性 真核生物Trn-ai-Met。 2。tRNA/氨基酰基-TRNA合成酶：尝试准备X射线质量时 将进行复杂的计算机图形实验的晶体 用Met-tRNA合成酶模型“对接” tRNA-i-met的结构。 主要指南将是直接的Photocroslink和标记反应 这模拟了tRNA U8的瞬态反应 合成酶。 这些“ tethers”表明tRNA的核苷酸和 合成酶的氨基酸在彼此接触 复合物的分子界面。 3。细菌抑制：TRP阻遏物/操作员系统：最近 确定的TRP阻遏物的原子模型将得到完善并比较 到无活跃的AporePressor的高分辨率结构（无结合 色氨酸）和非活动伪压剂（Trytophan取代了 目前正在确定的竞争对抗者） 晶体学上。 努力将继续改善结晶 阻遏物和操作员的复合物。 X射线衍射分析将 准备合适的晶体时继续进行。 这些晶体结构为 以及来自其他实验室的突变分析应有助于开发 通过调节的序列特异性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。 将检查未来结构研究的新调节系统， 包括; （1）结合和修饰的红霉素抗性蛋白 核糖体RNA中的特定残基，（2）单体RNA T7噬菌体家族的聚合酶/启动子系统和（3） 哺乳动物细胞系的类固醇受体/增强剂系统。