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，在嘌呤、氨基酸和维生素生物合成等基本代谢途径的调节中起关键作用。核开关使用多种不同的机制来调节配体结合时的基因表达，包括转录终止的调节、翻译抑制和自切割。从对核糖体开关作用机制的少数研究来看，配体结合动力学和RNA转录动力学在基因表达调控中起着重要作用。本提案的具体目的是：1)表征协同甘氨酸核糖开关的作用机制；2)构建核糖开关RNA折叠的动力学模型。通过一系列生物物理测量，将测量甘氨酸核糖开关的配体结合动力学，并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如何不同。此外，合作行为的序列决定因素将通过针对两个甘氨酸结合基序之间的系统发育上保守的连接体来探索。基于系统发育和热力学分析，所有的核开关都存在二级结构模型。然而，大多数检测到的核开关具有缓慢的配体结合动力学，表明诱导配合机制。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。预计一些转录中间体允许比其他更快的折叠。预计这种建模可能会导致更好的工程RNA基因调控元件的计算设计策略的发展。由于它们广泛存在于原核生物中并调节必要的代谢过程，核糖开关成为抗菌剂的新靶点。此外，核糖开关是为合成生物学应用而设计的RNA元件的天然（通常也是更好的）例子。了解核开关的作用机制也将有助于更好地设计工程化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.