Control of Bacterial Nucleoid Structure

细菌核结构的控制

• 批准号: 8036899
• 负责人: JOHN W SHRIVER
• 金额: $ 44.13万
• 依托单位: UNIVERSITY OF ALABAMA IN HUNTSVILLE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8036899
• 关键词: Control; Bacterial; Nucleoid; Structure

DESCRIPTION (provided by applicant): Our long-term goal is to provide an accurate description of the role of IHF, HU, and associated proteins in controlling bacterial nucleoid structure and gene expression. Changes in these proteins are linked to changes in the metabolism, growth and virulence through changes in both nucleoid structure and gene regulation. In this proposal we focus on the relationship between structure, solution states, and DNA binding by IHF and related proteins. Although IHF is commonly viewed as a sequence-specific DNA binding protein, and HU a non-sequence specific homolog, it is becoming increasingly clear that this view is too simplistic. We know a great deal about the structures of these proteins, both free in solution and bound to DNA, but our knowledge of their behavior in solution and the effect of solvent conditions and accessory proteins on DNA binding is limited and perplexing. In particular, the DNA binding energetics of IHF are extraordinary and it is currently believed that this is due to a large number of "masked" surface salt bridges which control the salt and temperature dependence of DNA binding and bending. This is thought by some workers to be a "signature" for DNA wrapping surfaces and it has been proposed to be of general significance for a number of DNA binding proteins. We propose that this interpretation is incorrect because it ignores important properties of IHF structure and solution behavior. We hypothesize that the unusual DNA binding energetics of IHF and the salt/temperature dependence of DNA binding are linked to marginal stability and intrinsic disorder of IHF, and the induced folding that accompanies binding. In order to test this and build a firm foundation for future studies of the assembly and control of higher order structures involving IHF and related proteins, we propose a search for surface salt bridges in these proteins using NMR, along with a systematic characterization of protein solution properties and their linkage to DNA binding using ITC, CD, DSC, and analytical ultracentrifugation. To investigate the generality of this proposal, we will use comparative studies of homologous proteins with a range of stabilities and DNA binding affinities to unambiguously define the linkage of structure and binding. This work will provide a description of IHF/HU solution properties and states which are essential to providing a rigorously correct understanding of the control and relative affinities of specific and non-specific binding by the IHF/HU protein family. PUBLIC HEALTH RELEVANCE: The results of this work will provide an accurate, quantitative description of the solution states of a family of proteins which play a central role in many protein-DNA interactions important in bacterial growth and human disease. This work is a necessary first step to understanding the role of these proteins in controlling bacteria gene expression, metabolism, growth and virulence through changes in both DNA packaging and gene regulation.

描述（由申请人提供）：我们的长期目标是准确描述IHF、HU和相关蛋白在控制细菌类核结构和基因表达中的作用。这些蛋白质的变化通过类核结构和基因调控的变化与代谢、生长和毒力的变化相关。在这个建议中，我们专注于IHF和相关蛋白质的结构，溶液状态和DNA结合之间的关系。虽然IHF通常被认为是一种序列特异性DNA结合蛋白，而HU是一种非序列特异性同源物，但越来越清楚的是，这种观点过于简单化。我们对这些蛋白质的结构了解很多，无论是在溶液中还是与DNA结合，但我们对它们在溶液中的行为以及溶剂条件和辅助蛋白质对DNA结合的影响的了解是有限且令人困惑的。特别是，IHF的DNA结合能是非凡的，目前认为这是由于大量的“掩蔽”表面盐桥，其控制DNA结合和弯曲的盐和温度依赖性。一些研究者认为这是DNA包裹表面的“特征”，并且已经提出它对许多DNA结合蛋白具有普遍意义。我们认为这种解释是不正确的，因为它忽略了IHF结构和溶液行为的重要性质。我们假设，不寻常的DNA结合能的IHF和DNA结合的盐/温度依赖性与边缘稳定性和内在的IHF的紊乱，以及诱导的折叠，伴随着绑定。为了测试这一点，并建立一个坚实的基础，为未来的研究，涉及IHF和相关蛋白质的组装和控制的高阶结构，我们提出了一个搜索表面盐桥在这些蛋白质中使用NMR，沿着系统的表征蛋白质溶液的性质和它们的链接到DNA结合使用ITC，CD，DSC，和分析超离心。为了研究这一建议的一般性，我们将使用同源蛋白质的比较研究，具有一系列的稳定性和DNA结合亲和力，明确定义的结构和结合的联系。这项工作将提供一个描述IHF/HU解决方案的性质和状态，这是必不可少的提供一个严格正确的理解控制和相对亲和力的特异性和非特异性结合的IHF/HU蛋白质家族。 公共卫生关系：这项工作的结果将提供一个准确的，定量的描述一个家庭的蛋白质的溶液状态，发挥了核心作用，在许多蛋白质-DNA相互作用的重要细菌生长和人类疾病。这项工作是了解这些蛋白质通过DNA包装和基因调控的变化在控制细菌基因表达、代谢、生长和毒力方面的作用的必要的第一步。