EXPERIMENTAL PHASING OF A METABOLITE SENSING RIBOSWITCH

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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.