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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 分子氧是有氧生活的基本要求，但氧化应激会损害大分子，如果不加控制，可能是致命的。到目前为止，调控转录响应于氧化还原状态的变构构象变化已经可视化在少数情况下，如果有的话。该研究的长期目标是阐明响应细胞内氧化还原状态调节DNA结合的新的和必要的变构机制。 在革兰氏阳性细菌中，氧化还原敏感抑制子（雷克斯）采用区分必需辅因子NAD的还原和氧化状态的优雅方法。雷克斯结合DNA靶标，并在氧化NAD+存在下抑制呼吸基因的转录，但在还原型NADH水平异常升高时授权转录。该实验室先前确定了雷克斯/NADH复合物的X射线结构。虽然雷克斯/NADH结构提供了一个重要的观点，雷克斯的状态，不能识别DNA，变构反应的基本机制雷克斯的NADH/NAD+仍然是未知的。需要了解雷克斯/NAD+/DNA复合物的结构，以了解雷克斯如何重新排列以识别DNA，以及NAD+/NADH之间的微小差异如何触发这种转变。 为了实现这一目标，已经获得了雷克斯/NAD+/DNA共晶体。从溶解晶体的A280：A260比率验证DNA和蛋白质两者的存在。虽然晶体的分辨率超过3，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MAD定相与溴化DNA，并提供额外的相位信息，由一个单一的SeMet每个单体。基于雷克斯/NADH和DNA的不相容形状，雷克斯可能在NADH/NAD+交换后经历大的构象变化。假设的雷克斯的构象重排将揭示一个新的模式，转录调控响应NAD+和NADH之间的最小差异。