REDOX-SENSING REPRESSOR

氧化还原传感抑制子

• 批准号: 7726275
• 负责人: CLARA KIELKOPF
• 金额: $ 1.04万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-18 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7726275
• 关键词: Adopted; Aerobic; Complex; Computer Retrieval of Information on Scientific Projects Database; DNA; DNA Binding; DNA Sequence Rearrangement; Data; Funding; Genes; Genetic Transcription; Goals; Gram-Positive Bacteria; Grant; Housing; Institution; L-Selenomethionine; Laboratories; Life; Molecular; NADH; Nicotinamide adenine dinucleotide; Oxidation-Reduction; Oxidative Stress; Oxygen; Phase; Proteins; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Shapes; Source; Structure; Transcriptional Regulation; United States National Institutes of Health; base; cofactor; macromolecule; monomer; novel; respiratory; response

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Molecular oxygen is a fundamental requirement for aerobic life, yet oxidative stress damages macromolecules and can be lethal if unchecked. Thus far, allosteric conformational changes that regulate transcription in response to the redox state have been visualized in few, if any cases. The long term goal of the proposed research is to elucidate novel and essential allosteric mechanisms that regulate DNA-binding in response to the intracellular redox state. In Gram-positive bacteria, the redox-sensing repressor (Rex) adopts the elegant approach of distinguishing the reduced and oxidized states of the essential cofactor, NAD. Rex binds the DNA target and represses transcription of respiratory genes in the presence of oxidized NAD+, yet authorizes transcription when reduced NADH levels abnormally rise. This laboratory previously determined the X-ray structure of the Rex/NADH complex. Although the Rex/NADH structure provided one important view of the state of Rex that is unable to recognize DNA, the fundamental mechanism of the allosteric response of Rex to NADH/NAD+ remains unknown. The structure of the Rex/NAD+/DNA complex is needed to understand how Rex rearranges to recognize DNA, and how the slight differences between NAD+/NADH trigger this transition. Towards this goal, Rex/NAD+/DNA cocrystals have been obtained. The presence of both DNA and protein was verified from the A280:A260 ratio of dissolved crystals. Although the crystals diffract beyond 3¿¿¿ resolution, a 300¿¿¿ c-axis limits our ability to collect complete, high-resolution data in-house. The structure will be solved by MIR or MAD phasing with brominated-DNA, and additional phase information provided by a single SeMet per monomer. Based on the incompatible shapes of Rex/NADH and DNA, Rex is likely to undergo a large conformational change following NADH/NAD+ exchange. The hypothesized conformational rearrangement of Rex would reveal a new paradigm for transcriptional regulation in response to the minimal differences between NAD+ and NADH.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 分子氧是有氧寿命的基本要求，但氧化应激会损害大分子，如果未检查，可能是致命的。如果有的话，已经对氧化还原状态的转录进行调节的变构会议变化已被可视化。拟议的研究的长期目标是阐明针对细胞内氧化还原态的新型和必不可少的变构机制，这些机制调节了DNA结合。 在革兰氏阳性细菌中，氧化还原感应复制器（REX）采用了优雅的方法，可以区分基本辅助因子NAD的降低和氧化状态。 Rex结合DNA靶标，并反映在氧化NAD+存在下呼吸基因的转录，但在降低NADH水平的绝对上升时授权转录。该实验室先前确定了REX/NADH复合物的X射线结构。尽管REX/NADH结构提供了无法识别DNA状态的重要看法，但REX对NADH/NAD+的变构响应的基本机制仍然未知。需要REX/NAD+/DNA复合物的结构，以了解Rex如何重新排列DNA，以及NAD+/NADH之间的轻微差异如何触发此转变。 为了实现这一目标，已经获得了REX/NAD+/DNA共晶。从A280：A260的溶解晶体比例验证了DNA和蛋白质的存在。尽管晶体衍射超出了3€€的分辨率，但300€»c轴限制了我们在内部收集完整的高分辨率数据的能力。该结构将通过mir或用溴化-DNA的mir或mad逐步求解，以及每月单个SEMET提供的其他阶段信息。基于REX/NADH和DNA的不兼容的形状，REX可能会在NADH/NAD+交换后发生巨大的构象变化。 REX的假设构象重排将揭示针对NAD+和NADH之间最小差异的转录调节的新范式。