EXPERIMENTAL PHASING OF A METABOLITE SENSING RIBOSWITCH

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 核糖开关是位于细菌基因5‘非翻译区的非蛋白质编码的RNA元件。这些元件可以通过促进替代结构来控制基因的表达，这些结构屏蔽了代谢物结合反应中的转录或翻译信号。之前，我的实验室结晶了一种小的、对代谢物敏感的核糖开关，它能结合奎宁前体。目前，这种结构尚不清楚，代表了一个新的RNA折叠。利用国际象棋，我们证明了天然晶体的衍射率为3.2埃。也制备了单一的碘衍生物，但源的低通量排除了对异常信号的检测。在这项建议中，我们将收集额外的5-碘-尿嘧啶晶体(即新的位置)的数据，以解决双峰相分布。此外，我们建议：(I)收集更高分辨率的天然数据；(Ii)收集包括异常信号在内的现有重原子的更高分辨率的导数数据；以及(Iii)收集存在不同代谢物的数据集，以了解配体专一性的基础。确定这种核糖开关的结构是当务之急。

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.