Characterization of Natural mRNA Genetic Switches that Bind Metabolites

描述(申请人提供)：核糖开关是天然的顺式作用的RNA遗传调节元件。它们结合代谢物，并在包括嘌呤、氨基酸和维生素生物合成在内的基本代谢途径的调节中发挥关键作用。核糖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、翻译抑制和自我切割的调节。从对核糖开关作用机制的研究来看，配基结合动力学和RNA转录在基因表达调控中起着重要作用。这一建议的具体目的是1)表征合作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，并将探索转录速度如何影响开关的行为。将检查开关中预期的转录暂停位置，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有何不同。此外，将通过靶向两个甘氨酸结合基序之间的系统发育保守的连接子来探索合作行为的序列决定因素。根据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，所研究的大多数核糖开关具有缓慢的配体结合动力学，表明存在诱导FIT机制。将使用现有的RNA二级结构折叠模拟来检查配体结合所需的二级结构构象的动力学可及性。据预计，一些转录中间体比其他中间体折叠得更快。预计这种建模可能会导致开发出更好的工程RNA基因调控元件的计算设计策略。由于核糖开关广泛存在于原核生物中，并调节必要的代谢过程，因此成为抗菌药物的新靶点。此外，核糖开关是为合成生物学应用而设计的RNA元件的自然(通常是更好的)例子。了解核糖开关的作用机制也将有助于更好地设计工程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.