EM PHYSICAL STUDIES OF DNA BINDING PROTEIN COMPLEXES

DNA 结合蛋白复合物的电子显微镜物理研究

• 批准号: 3285010
• 负责人: CARLA W GRAY
• 金额: $ 5.98万
• 依托单位: UNIVERSITY OF TEXAS DALLAS
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-01-01 至 1987-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3285010
• 关键词: bacterial virus; chemical binding; chemical bond; circular dichroism; conformation; electron microscopy; ionic strengths; nucleic acid reconstitution; nucleic acid sequence; nucleic acid structure; polynucleotides; virus DNA; virus RNA; virus genetics

High resolution electron microscopy is used in physical studies of protein-nucleic acid complexes in two new areas. (1) The first is a comparison of complexes formed by the analogous but physically dissimilar DNA binding proteins of the filamentous viruses fd and Pfl; the fd gene 5 protein is one of the most intensively studied "model" DNA binding proteins presently available, and the Pfl analogue was recently discovered. How do these dissimilar proteins perform similar functions? The detailed three-dimensional structure of these helical complexes will be investigated, including conformational shifts, topological rearrangements, dissociation and reassembly, and a possible disk-to-helix transition accompanying a shift from an n=3 to an n=4 binding mode. Models will be developed for the arrangement of proteins and DNA in these complexes and for protein-DNA interactions as they relate to biological function. (2) The second area of study involves the use of the fd gene 5 protein as a "coating" on polynucleotides to amplify and directly visualize polynucleotide helical structure, an approach which preliminary investigations indicate should be feasible. This may provide a means for direct confirmation of unusual structures such as the left-handed "Z" or "loopout" forms of DNA, and for correlation with spectral data indicating the existence of these DNA forms in solution. The methodology for this work primarily involves the quantitative analysis of negatively-stained nucleoprotein structures visualized in an electron microscope at very high magnifications, using tilting/rotation in a goniometer stage to derive three-dimensional structural information. Circular dichroism (CD) spectroscopy is used for protein binding titrations and to correlate solution structural data (e.g., Z-DNA spectrum) with structures analyzed by microscopy. This work is health-related in that it is (1) a fundamental study of one of the best available examples of a class of proteins (helix-destabilizing proteins) that are likely to be universally involved in the processes of DNA replication, recombination, and repair, and (2) a study of unusual DNA structures that may enter into, for example, transcriptional control. Knowledge of these elemental biological processes is essential to understanding cellular function in health and disease.

高分辨率电子显微镜用于物理研究 两个新领域的蛋白质-核酸复合物。 (1) 第一个是 由类似但物理上不同的物质形成的复合物的比较 丝状病毒fd和Pfl的DNA结合蛋白； fd基因5 蛋白质是研究最深入的“模型”DNA 结合蛋白之一 目前可用，并且最近发现了 Pfl 类似物。 怎么办 这些不同的蛋白质执行相似的功能？ 详细的 这些螺旋复合物的三维结构将是 研究，包括构象转变、拓扑重排、 解离和重组，以及可能的盘到螺旋的转变 伴随着从n=3到n=4结合模式的转变。 型号将是 为这些复合物中蛋白质和 DNA 的排列而开发 蛋白质-DNA 相互作用，因为它们与生物功能有关。 (2) 第二个研究领域涉及使用 fd 基因 5 蛋白作为 “涂层”在多核苷酸上以放大并直接可视化 多核苷酸螺旋结构，一种初步 调查表明应该是可行的。 这可能提供一种手段 直接确认不寻常的结构，例如左手“Z”或 DNA 的“循环”形式，以及与光谱数据的相关性表明 这些DNA形式在溶液中的存在。 这项工作的方法主要涉及定量分析 在电子中可视化的负染色核蛋白结构 显微镜在非常高的放大倍数下，使用倾斜/旋转 测角器阶段以获得三维结构信息。 圆二色性 (CD) 光谱用于蛋白质结合滴定 并将溶液结构数据（例如 Z-DNA 谱）与 通过显微镜分析结构。 这项工作与健康相关，因为它是 (1) 以下一项的基础研究： 一类蛋白质的最佳可用例子（螺旋不稳定 蛋白质）可能普遍参与以下过程 DNA 复制、重组和修复，以及 (2) 对异常 DNA 的研究 例如，可能进入转录控制的结构。 了解这些基本的生物过程对于 了解健康和疾病中的细胞功能。