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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

基本信息

批准号：
3285011
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
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-01-01 至 1988-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3285011
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

项目摘要

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蛋白质-核酸复合体在两个新领域。(1)第一个是相似但物理不同形成的络合物的比较丝状病毒Fd和Pf1的DNA结合蛋白；Fd基因5 蛋白质是研究最深入的dna结合蛋白之一。目前可用，PFL类似物是最近发现的。做什么这些不同的蛋白质有相似的功能吗？详细的这些螺旋络合物的三维结构将是研究，包括构象移动，拓扑重排，解离和重组，以及可能的盘状到螺旋的转变伴随着从n=3到n=4绑定模式的转变。模特们将成为为蛋白质和DNA在这些复合体中的排列而开发的用于蛋白质-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将fd基因5蛋白用作 “包被”在多核苷酸上进行扩增和直接可视化多核苷酸螺旋结构，一种初步的调查表明，这应该是可行的。这可能会提供一种手段直接确认不寻常的结构，如左撇子“Z”或 DNA的“环路”形式，以及与光谱数据的相关性这些DNA形式在溶液中的存在。这项工作的方法论主要涉及定量分析。电子中可见的负染核蛋白结构显微镜在非常高的放大倍数下，使用倾斜/旋转测角仪阶段，以获取三维结构信息。圆二色谱(CD)用于蛋白质结合滴定并将溶液结构数据(例如，Z-DNA谱)与显微镜下分析结构。这项工作是与健康有关的，因为它是(1)对以下内容之一的基础研究一类蛋白质(破坏螺旋稳定)的最佳可用实例蛋白质)，这些蛋白质可能普遍参与 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。对这些基本生物过程的了解对于了解细胞在健康和疾病中的功能。