Structural and temporal analyses of the folding landscape of the B12 riboswitch

描述（由申请人提供）：核糖开关是在mRNA转录物的52-非翻译区中发现的RNA调节元件，其调节基因表达。特定的小代谢物与核糖开关的适体结构域的结合会引起结构变化，从而发出信号，在表达平台中形成替代结构，从而影响基因表达。核糖开关首先在细菌中发现，它控制着致病菌中许多重要的和必需的基因。然而，适体结构域内的构象变化与下游结构形成的耦合机制尚不清楚。这项工作的目标是使用标准的生物化学技术来表征B12核糖开关的适体结构域内发生的空间和时间结构变化。在目标1中，将使用化学探测技术研究适体结构域的局部结构变化，该技术将突出镁和腺苷钴胺素（adenosylcobalamin，AIBCbl）促进稳定结构的重要性。此外，野生型和突变型B12核糖开关的溶液X射线散射将有助于确定溶液中的螺旋排列和包装以及在B12 Cbl结合后的变化。在目标2中，时间分辨的羟基自由基足迹法将研究在镁存在下适体折叠的速率，以及是否有β-Cbl辅助该过程。使用共转录时间分辨足迹法，将在天然转录的时间范围内研究适体结构域折叠的重要性。最后，为了理解配体结合如何被传递到表达平台，将确定具有和不具有p53 Cbl的整个核糖开关的RNA折叠速率。这些实验的结果将增加核糖开关结构形成的一般知识以及开始剖析基因调控的机制。 公共卫生相关性：核糖开关是主要通过小代谢物依赖方式控制细菌细胞中基因表达的RNA元件。随着越来越多的细菌菌株对目前的抗菌药物产生耐药性，对核糖开关如何工作的更深入了解将为抗菌治疗开辟新的可能性。

DESCRIPTION (provided by applicant): Riboswitches are elements of RNA regulation found in the 52-untranslated region of mRNA transcripts that regulate gene expression. Binding of a specific small metabolite to the aptamer domain of the riboswitch causes structural changes that signal the formation of an alternative structure in the expression platform which in turns affects gene expression. Primarily found in bacteria, riboswitches have been shown to control many important and essential genes in virulent pathogens. However, the mechanism of coupling conformational changes within the aptamer domain to downstream structure formation is unknown. The goal of this work is to characterize the spatial and temporal structural changes that occur within the aptamer domain of the B12 riboswitch using standard biochemical techniques. In Aim 1, the local structural changes to the aptamer domain will be investigated using chemical probing techniques that will highlight the importance of magnesium and adenosylcobalamin (AdoCbl) to promote a stable structure. Also, solution X-ray scattering of wild type and mutant B12 riboswitches will help define helical arrangement and packaging in solution and changes upon AdoCbl binding. In Aim 2, time-resolved hydroxyl radical footprinting will investigate the rate of aptamer folding in the presence of magnesium and if AdoCbl aids this process. Using co-transcriptional time-resolved footprinting, the importance of the aptamer domain folding will be investigated under the timeframe of native transcription. Finally, to understand how ligand binding is conveyed to the expression platform, rates of RNA folding of the entire riboswitch with and without AdoCbl will be determined. The results of these experiments will increase the general knowledge of riboswitch structure formation as well as begin to dissect the mechanism of gene regulation. PUBLIC HEALTH RELEVANCE: Riboswitches are RNA elements that control gene expression primarily in bacterial cells through a small metabolite-dependent fashion. As more bacterial strains become resistant to current antimicrobial drugs, a deeper understanding of how riboswitches work will open up new possibilities of antimicrobial therapeutics.