Understanding transcriptional silencing & anti-silencing mechanisms in Shigella

了解转录沉默

• 批准号: 8575890
• 负责人: HELEN J WING
• 金额: $ 43.15万
• 依托单位: UNIVERSITY OF NEVADA LAS VEGAS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8575890
• 关键词: Anti-Bacterial Agents; Bacteria; Bacterial Genes; Bacterial Physiology; Binding; Binding Sites; Biological Models; Cells; Coin; Complex; DNA; DNA Packaging; DNA Sequence; DNA-Directed RNA Polymerase; DNase-I Footprinting; Development; Drug Targeting; Enteral; Epigenetic Process; Eukaryotic Cell; Event; Figs - dietary; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genetic; Genetic Transcription; Goals; Histones; In Vitro; Lead; Learning; Maps; Mediating; Microbial Biofilms; Molecular; Mutate; Nevada; Plasmids; Play; Process; Progress Reports; Proteins; Reporting; Repression; Role; Shigella; Shigella flexneri; Site; Stress; Superhelical DNA; System; Testing; Transcription Initiation; Transcription Initiation Site; Transcriptional Regulation; Universities; Ursidae Family; Virulence; Work; gene repression; graduate student; improved; in vivo; innovation; novel; pathogen; promoter; protein structure; public health relevance; research study; tool; transcription factor; undergraduate student

DESCRIPTION (provided by applicant): Summary The nucleoid structuring protein H-NS plays important roles in the transcriptional silencing of many bacterial genes. Proteins that alleviate this silencing are central to bacterial physiology and control many processes including virulence gene expression, biofilm formation and bacterial adaptation to stress. Despite recent advances, transcriptional silencing and anti-silencing mechanisms remain poorly understood. An improved understanding of these processes could lead to the discovery of novel drug targets and/or development of new antibacterials. Here we propose to use the bacterial pathogen Shigella as a model system to study this mechanism of transcriptional regulation. The icsP gene, which is carried by the Shigella virulence plasmid, is transcriptionally repressed by H-NS and derepressed by the transcription factor VirB. Two sites (essential sites), located over 1 kb upstream of the icsP transcription start site, match the reported VirB binding sites and are required for transcriptiona anti-silencing. Our long term goal is to understand how VirB alleviates H-NS- mediated repression of virulence genes in Shigella flexneri. The overarching hypothesis of this project is "H- NS-mediated transcriptional repression of the Shigella icsP promoter is relieved when VirB binds to the essential sites, oligomerizes along DNA and triggers localized changes in DNA supercoiling". Our Specific Aims are: 1) to determine the role that the essential sites play in Vir oligomerization along DNA, 2) to determine the role that VirB-induced changes in plasmid supercoiling play in transcriptional anti-silencing by VirB, and 3) to determine whether the essential sites are sufficient for VirB binding in vivo; a key step in VirB- dependent transcriptional anti-silencing. The work we propose challenges classical views of the system we study, and a new genetic tool will be created to allow VirB binding to be assessed in vivo. Our project can therefore be considered conceptually and methodologically innovative. All experiments proposed in this application have been carefully selected so they are suitable for the graduate and undergraduate students at the University of Nevada, Las Vegas. By testing our overarching hypothesis we will learn more about the VirB protein, which can be considered the "molecular switch" that controls Shigella virulence, its interplay with the nucleoid structurin protein H-NS and the mechanisms that allow transcriptionally silenced genes to become active in Shigella and possibly other bacteria.

描述（由申请人提供）： 摘要核苷结构蛋白H-NS在许多细菌基因的转录沉默中起重要作用。减轻这种沉默的蛋白质是细菌生理学的核心，并控制许多过程，包括毒力基因表达，生物膜形成和细菌适应压力。尽管最近进步，但转录沉默和反沉降机制仍然鲜为人知。对这些过程的改进理解可能会导致发现新的药物靶标和/或新抗菌物的发展。在这里，我们建议将细菌病原体志贺氏菌作为模型系统，以研究这种转录调控机制。由志贺氏菌毒力质粒携带的ICSP基因被H-NS抑制，并由转录因子ViRB抑制。位于ICSP转录起始位点上游1 kb以上的两个位点（必需位点）与报道的VIRB结合位点匹配，并且是转录抗沉积所必需的。我们的长期目标是了解VIRB如何减轻志贺氏菌Flexneri中毒力基因的H-NS介导的抑制作用。该项目的总体假设是“当VirB结合到必需位点时，H- NS介导的Shigella ICSP启动子的转录抑制作用会缓解，沿DNA寡聚并触发DNA超架架子的局部变化”。我们的具体目的是：1）确定基本位点在沿DNA的ViR寡聚中发挥的作用，2）确定VIRB诱导的质粒超凝结变化在virb转录抗沉积中的作用，以及3）确定基本位点是否足以使VIRB与VirB Intivo In Vivo Intivo In VirB结合；依赖于VIRB的转录反沉降的关键步骤。我们提出的对我们研究系统的经典观点的挑战，并将创建一种新的遗传工具，以允许在体内评估VIRB结合。因此，我们的项目可以在概念和方法上被视为创新。该应用程序中提出的所有实验均已仔细选择，因此它们适合内华达大学内华达大学的研究生和本科生。通过测试我们的总体假设，我们将了解有关VIRB蛋白的更多信息，该蛋白可以被视为控制志贺氏菌毒力的“分子开关”，它与核苷结构蛋白蛋白H-NS的相互作用以及允许转录沉默的基因在Shigella中变得活跃的机制以及其他可能的其他细菌。