THE MECHANISM OF ACTION OF THE TRANSCRIPTIONAL REPRESSOR NMRA-DESCRIPTION

转录抑制子 NMRA 的作用机制-描述

• 批准号: 8359746
• 负责人: RICHARD D TODD
• 金额: $ 6.05万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8359746
• 关键词: Affect; Area; Biomedical Research; Deacetylase; Detection; Enzymes; Funding; Genes; Grant; Kansas; Mediating; Metabolic; Molecular Genetics; Molecular Mechanisms of Action; Mutation; National Center for Research Resources; Nitrogen; Nutritional; Phenotype; Principal Investigator; Proteins; Proteomics; Repression; Research; Research Infrastructure; Resources; Sequence Determination; Source; Starvation; System; Transcription Repressor/Corepressor; United States National Institutes of Health; cost; genetic analysis; mutant; novel; overexpression; protein protein interaction; response; transcription factor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This research aims to determine the molecular mechanism of action of the A. nidulans transcriptional corepressor NmrA, a key component of an important regulatory system governing nutritional response. NmrA represses during nitrogen sufficiency the activity of the AreA GATA transcription factor. AreA activates expression of nitrogen metabolic genes during nitrogen limitation or nitrogen starvation. NmrA is thought to inhibit AreA activity by direct protein-protein interaction but the mechanism of NmrA action is not understood. This proposal will investigate how the corepressor NmrA regulates AreA activity. The key aims are (1) to establish whether NmrA acts as a regulatory NAD-dependent protein deacetylase enzyme, (2) to identify NmrA interactors by proteomics, and (3) to identify a new gene involved in NmrA-mediated repression. Objective 1: Does NmrA mediate repression by functioning as a regulatory enzyme? 1.1 Detection of NAD-dependent deacetylase activity using the Fluor-de-lys" substrate. Objective 2: Identification of proteins that interact with the NmrA repressor using a proteomics approach. 2.1 Biacore capture of proteins that interact with NmrA. Objective 3: Characterization of a suppressor of an NmrA overexpression phenotype. 3.1 Determination of the sequence change in a mutant affected in a novel gene affecting NmrA function by massively parallel sequencing. 3.2 Functional confirmation of mutations identified in the unknown gene. 3.3 Initial molecular genetic analysis of the new player in NmrA-mediated repression.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 子项目的主要研究者可能是由其他来源提供的， 包括其他NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 本研究旨在确定A. nidulans转录辅抑制因子NmrA是一个重要的营养反应调控系统的关键组成部分。NmrA在氮充足期间抑制AreA加塔转录因子的活性。AreA在氮限制或氮饥饿期间激活氮代谢基因的表达。NmrA被认为通过直接的蛋白质-蛋白质相互作用抑制AreA活性，但NmrA作用的机制尚不清楚。该提案将研究辅阻遏物NmrA如何调节AreA活性。主要目的是（1）确定NmrA是否作为一种调节NAD依赖性蛋白脱乙酰酶，（2）通过蛋白质组学鉴定NmrA相互作用物，（3）鉴定一种参与NmrA介导的抑制的新基因。 目的1：NmrA是否通过调节酶的功能介导阻遏？ 1.1 使用Fluor-de-lys底物检测NAD依赖性脱乙酰酶活性。 目的2：使用蛋白质组学方法鉴定与NmrA阻遏物相互作用的蛋白质。 2.1 Biacore捕获与NmrA相互作用的蛋白质。 目的3：表征NmrA过表达表型的抑制子。 3.1 确定在新的基因影响中受影响的突变体中的序列变化， NmrA功能通过大规模平行测序。 3.2 在未知基因中鉴定的突变的功能确认。 3.3 NmrA介导的阻遏中新参与者的初步分子遗传学分析。