Characterization of Natural mRNA Genetic Switches that Bind Metabolites

描述（由申请人提供）：核糖开关是天然的顺式作用RNA遗传调控元件。它们结合代谢物并在调节包括嘌呤、氨基酸和维生素生物合成的基本代谢途径中具有关键作用。核糖开关使用多种不同的机制来调节配体结合后的基因表达，包括转录终止、翻译抑制和自切割的调节。从核糖开关作用机制的少数研究来看，配体结合和RNA转录的动力学在基因表达调控中起着重要作用。本研究的具体目标是：1）表征甘氨酸核糖开关的协同作用机制; 2）构建核糖开关RNA折叠的动力学模型。通过一系列的生物物理测量，甘氨酸核糖开关的配体结合动力学将被测量，并将探讨转录速率如何影响开关的行为。将检查开关内的预期转录暂停位点，以进一步研究与整个开关相比，离散中间体的配体结合亲和力可能如何不同。此外，合作行为的序列决定因素将通过靶向两个甘氨酸结合基序之间的遗传学保守的接头来探索。基于系统发育和热力学分析，所有的核糖开关都存在二级结构模型。然而，大多数核糖开关检查有缓慢的配体结合动力学，表明诱导适合机制。配体结合所需的二级结构构象的动力学可及性将使用现有的RNA二级结构折叠模拟进行检查。预期一些转录中间体允许比其它中间体更快的折叠。预计这种建模可能会导致更好的计算设计策略的工程RNA基因调控元件的发展。由于核糖开关在原核生物中的广泛存在和重要代谢过程的调节，核糖开关代表了抗菌剂的新靶点。此外，核糖开关是用于合成生物学应用的RNA元件的天然（并且通常更好）例子。理解核糖开关的作用机制也将导致更好地设计工程RNA元件。

DESCRIPTION (provided by applicant): Riboswitches are natural cis-acting RNA genetic regulatory elements. They bind metabolites and have critical roles in the regulation of basic metabolic pathways including purine, amino acid and vitamin biosynthesis. Riboswitches use a variety of different mechanisms to regulate gene expression upon ligand binding including modulation of transcription termination, translation suppression, and self-cleavage. From the few studies of riboswitch mechanism of action, the kinetics of ligand binding and RNA transcription play an important role in the regulation of gene expression. The specific aims of this proposal are to 1) characterize the cooperative glycine riboswitch mechanism of action and 2) construct kinetic models of riboswitch RNA folding. Through a series of biophysical measurements, the ligand binding kinetics of the glycine riboswitch will be measured and how the rate of transcription may affect the behavior of the switch will be explored. Prospective transcriptional pause sites within the switch will be examined to further study how ligand binding affinity may differ for discreet intermediates compared with the entire switch. Additionally, the sequence determinants for the cooperative behavior will be explored by targeting the phylogenetically conserved linker between the two glycine binding motifs. Secondary structure models exist for all the riboswitches based on phylogenetic and thermodynamic analysis. However, most riboswitches examined have slow ligand binding kinetics indicating an induced fit mechanism. The kinetic accessibility of secondary structure conformations necessary for ligand binding will be examined using existing RNA secondary structure folding simulations. It is anticipated that some transcriptional intermediates allow faster folding than others. It is anticipated that this modeling may lead to the development of better computational design strategies for engineered RNA gene regulatory elements. Due to their widespread appearance in prokaryotes and regulation of essential metabolic processes, riboswitches represent new targets for antimicrobial agents. Additionally, riboswitches are natural (and often better) examples of RNA elements engineered for synthetic biology applications. Understanding riboswitch mechanism of action also will lead to better design of engineered RNA elements.