EXPERIMENTAL PHASING OF A METABOLITE SENSING RIBOSWITCH

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。 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，所述替代结构响应于代谢物结合而掩蔽转录或翻译信号。 以前，我的实验室结晶了一个小的，代谢物感应核糖开关，结合奎奥辛前体。 目前，这种结构是未知的，代表了一种新的RNA折叠。使用CHESS，我们证明了天然晶体的分辨率为3.2埃。还制备了单一的碘衍生物，但是源的低通量排除了异常信号的检测。 在本提案中，我们将收集额外的含5-碘-尿嘧啶晶体（即新位点）的数据，以解析双峰相分布。 此外，我们建议：（i）收集更高分辨率的天然数据;（ii）收集现有重原子的更高分辨率的衍生数据，包括异常信号;以及（iii）收集不同代谢物存在下的数据集，以了解配体特异性的基础。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.