THE MECHANISM OF ACTION OF THE TRANSCRIPTIONAL REPRESSOR NMRA-DESCRIPTION

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 这项研究旨在确定根结线虫转录辅阻遏子NmrA的分子作用机制，NmrA是调控营养反应的重要调控系统的关键组成部分。在氮充足期间，NmrA抑制区域GATA转录因子的活性。在氮素限制或氮素饥饿过程中，AREA激活氮代谢基因的表达。NmrA被认为通过蛋白质与蛋白质的直接相互作用来抑制区域活性，但其作用机制尚不清楚。这项提案将调查辅阻遏子NmrA如何调节区域活动。主要目的是(1)确定NmrA是否是NAD依赖的蛋白去乙酰基酶的调节性酶；(2)通过蛋白质组学鉴定NmrA的相互作用；(3)寻找与NmrA介导的抑制有关的新基因。 目标1：NmrA是否通过作为一种调节酶来调节抑制？ 1.1使用荧光素底物检测依赖NAD的脱乙酰酶活性。 目的2：用蛋白质组学方法鉴定与NmrA抑制物相互作用的蛋白质。 2.1与NmrA相互作用的蛋白质的Biacore捕获。 目的3：NmrA过表达表型的抑制子的特征。 3.1确定受影响的新基因中受影响的突变体的序列变化 NmrA通过大规模并行测序发挥作用。 3.2对未知基因中发现的突变进行功能确认。 3.3对NmrA介导的阻遏中新成员的初步分子遗传学分析。

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.