RUI: Defining the thermodynamics of MarR family transcription factor interactions with DNA

RUI：定义 MarR 家族转录因子与 DNA 相互作用的热力学

• 批准号: 1949109
• 负责人: Steven Wilkinson
• 金额: $ 31.13万
• 依托单位: California Polytechnic State University Foundation
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1949109&HistoricalAwards=false
• 关键词: RUI; Defining; thermodynamics; MarR; family

MarR family proteins are found in most bacterial species. These proteins bind very tightly to DNA and regulate the expression of genes that allow the bacterium to respond to various forms of environmental stress, including noxious chemicals and antibiotics. MarR proteins bind DNA with a common structural motif, yet each is specific for a particular sequence. The energetic basis for the affinity of these proteins for their target DNA sequences is not well understand. This project seeks to determine the structural components of MarR proteins that drive association with DNA. These studies will provide a detailed understanding of how MarR proteins interact with DNA, and are also expected to provide deeper insight into the energetics of sequence-specific protein-DNA interactions in general. In addition, these investigations will provide the first description of the roles of MarR proteins in regulating genes in the pathogenic bacterium Clostridium difficile and offer insight into this organism’s response to changing environmental conditions and stresses, including its resistance to antibiotics and antimicrobials. The project will be conducted at a primarily undergraduate institution and provide substantive training opportunities for undergraduate students who will participate in all phases of the project. This diverse group of students will conduct independent research using modern techniques in biochemistry, which will prepare them for advanced studies and careers in the STEM workforce. MarR proteins associate with DNA through a winged-helix-turn-helix (wHTH) motif, with nearly all interactions involving the wHTH “recognition helix” and “wing” which form base-specific contacts with the DNA major and minor grooves, respectively. However, the thermodynamics of these high-affinity associations is poorly understood. While DNA binding proteins have traditionally been classified as either major groove binders or minor groove binders, with each class being associated with a distinct thermodynamic signature, MarR proteins do not fall neatly into either category since they associate with DNA through extensive base-specific contacts in both grooves. The first objective of this project is to determine if there is a thermodynamic signature that characterizes MarR family binding interactions with DNA and to parse the energetic contributions to binding that stems from protein interactions with the major and minor grooves. More broadly, these findings will test the prevailing view that such interactions each have a characteristic thermodynamic signature. These objectives will be met through a systematic analysis of DNA binding thermodynamics involving six MarR homologs, including two which were previously structurally-defined, as well as four previously uncharacterized MarR homologs from the bacterium, Clostridium difficile.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

马尔R家族蛋白存在于大多数细菌物种中。这些蛋白质与DNA紧密结合，并调节基因的表达，使细菌能够对各种形式的环境压力做出反应，包括有毒化学物质和抗生素。马尔R蛋白结合DNA与一个共同的结构基序，但每一个是特定的序列。这些蛋白质对其靶DNA序列的亲和力的能量基础还不清楚。该项目旨在确定驱动与DNA结合的马尔R蛋白的结构组分。这些研究将提供一个详细的了解如何马尔蛋白质与DNA相互作用，也有望提供更深入的了解一般的序列特异性蛋白质-DNA相互作用的能量学。此外，这些研究将首次描述马尔R蛋白在调节致病性细菌艰难梭菌基因中的作用，并深入了解这种生物体对不断变化的环境条件和压力的反应，包括其对抗生素和抗菌剂的抗性。该项目将在一个主要的本科院校进行，并为将参加该项目所有阶段的本科生提供实质性培训机会。这个多元化的学生群体将使用现代生物化学技术进行独立研究，这将为他们在STEM劳动力中的高级学习和职业生涯做好准备。 马尔R蛋白通过翼螺旋转角螺旋（wHTH）基序与DNA结合，几乎所有的相互作用都涉及wHTH“识别螺旋”和“翼”，它们分别与DNA大沟和小沟形成碱基特异性接触。然而，这些高亲和力协会的热力学知之甚少。虽然DNA结合蛋白传统上被分类为大沟结合剂或小沟结合剂，每一类都与不同的热力学特征相关，但马尔蛋白并不完全属于任何一类，因为它们通过两个沟中广泛的碱基特异性接触与DNA结合。该项目的第一个目标是确定是否存在表征马尔R家族与DNA结合相互作用的热力学特征，并解析源于蛋白质与大沟和小沟相互作用的对结合的能量贡献。更广泛地说，这些发现将测试流行的观点，这种相互作用都有一个特征的热力学签名。这些目标将通过DNA结合热力学的系统分析，涉及六个马尔同系物，其中包括两个以前的结构定义，以及四个以前未表征的马尔同系物从细菌，艰难梭菌。这个奖项反映了美国国家科学基金会的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

项目成果

The structure of Deinococcus radiodurans transcriptional regulator HucR retold with the urate bound

耐辐射奇球菌转录调节因子 HucR 的结构与尿酸盐结合重述

• DOI: 10.1016/j.bbrc.2022.05.034
• 发表时间: 2022
• 期刊: Biochemical and Biophysical Research Communications
• 影响因子: 3.1
• 作者: Rho, SooHo;Jung, WeonSeok;Park, Jeong Kuk;Choi, Min Hee;Kim, MinJu;Kim, JooYoung;Byun, JiWon;Park, Taehyun;Lee, Byung Il;Wilkinson, Steven P.
• 通讯作者: Wilkinson, Steven P.