EXPERIMENTAL PHASING OF A METABOLITE SENSING RIBOSWITCH

代谢物传感核开关的实验定相

• 批准号: 7954488
• 负责人: Joseph E Wedekind
• 金额: $ 0.02万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954488
• 关键词: 5' Untranslated Regions; Bacterial Genes; Binding; Code; Computer Retrieval of Information on Scientific Projects Database; Data; Data Set; Detection; Elements; Funding; Gene Expression; Genetic Transcription; Grant; Institution; Iodine; Ligands; Masks; Phase; Proteins; RNA; RNA Folding; Research; Research Personnel; Resolution; Resources; Signal Transduction; Site; Source; Specificity; Structure; Translations; United States National Institutes of Health; Uracil; base; novel; response; structural biology; synchrotron radiation

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. Riboswitches are non-protein coding RNA elements at the 5'-untranslated regions of bacterial genes. These elements can control gene expression by promoting alternative structures that mask transcription or translation signals in response to metabolite binding. Previously my lab crystallized a small, metabolite-sensing riboswitch that binds queosine precursors. At present, this structure is unknown and represents a novel RNA fold. Using CHESS, we demonstrated that native crystals diffract to 3.2 Angstrom resolution. A single iodine derivative was also prepared, but the low flux of the source precluded detection of an anomalous signal. In this proposal, we will collect data on additional 5-iodo- uracil-containing crystals (i.e. new sites) to resolve the bimodal phase distribution. In addition, we propose: (i) to collect higher resolution native data; (ii) to collect higher resolution derivative data for our existing heavy atom including the anomalous signal; and (iii) to collect data sets in the presence of different metabolites in order to understand the basis for ligand specificity. Determining the structure of this riboswitch is an immediate priority.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 核糖开关是位于细菌基因 5'-非翻译区的非蛋白质编码 RNA 元件。 这些元件可以通过促进替代结构来控制基因表达，这些结构可响应代谢物结合而掩盖转录或翻译信号。 此前，我的实验室结晶了一种小型代谢物感应核糖开关，可结合奎奥辛前体。 目前，这种结构尚不清楚，它代表了一种新的 RNA 折叠。使用 CHESS，我们证明了天然晶体的衍射分辨率为 3.2 埃。还制备了单一碘衍生物，但源的低通量妨碍了异常信号的检测。 在本提案中，我们将收集有关其他含 5-碘尿嘧啶晶体（即新位点）的数据，以解决双峰相分布问题。 此外，我们建议：（i）收集更高分辨率的原生数据； (ii) 收集我们现有重原子的更高分辨率衍生数据，包括异常信号； (iii) 在存在不同代谢物的情况下收集数据集，以了解配体特异性的基础。确定该核糖开关的结构是当务之急。