In vitro and in vivo characterization of the cyclic-di-GMP riboswitch

 描述(申请人提供)：核糖开关是一种结构化的RNA，作为调节基因表达的一种机制，它会因特定的配体而发生显著的构象变化。除了几种蛋白质外，两种不同类型的c-di-GMP核糖开关还与环状二鸟苷一磷酸(c-di-GMP)第二信使途径有关，这是细菌中普遍存在的一种信号传递方式。对两类c-di-GMP核糖开关的晶体结构进行了解析，揭示了c-di-GMP识别的两种不同机制。这些核糖开关位于参与生物膜形成、细胞运动和毒力因子的基因的5‘-非翻译区。由于c-di-GMP核糖开关广泛存在于破伤风、炭疽、肉毒杆菌中毒和霍乱等细菌物种中，它们的特征具有直接的治疗应用。虽然这两类核糖开关的结构已经得到了广泛的表征，但在体内进行的实验很少。Strobel实验室合成了一系列c-di-GMP类似物，以区别研究两种c-di-GMP核糖开关的配体结合特性。这些配体中的几个也表现出对细胞内切割的抵抗力，是研究c-di-GMP核糖开关在细胞中行为的理想选择。这项建议试图确定c-di-GMP在细胞中的功能，并使用跨越生物化学、结构生物学、微生物学和遗传学的多学科方法来确定c-di-GMP核糖开关在细菌基因表达中的作用。

 DESCRIPTION (provided by applicant): Riboswitches are structured RNAs that undergo significant conformational changes in response to specific ligands as a mechanism to regulate gene expression. In addition to several proteins, two different classes of c-di-GMP riboswitches have been implicated in the cyclic diguanosine monophosphate (c-di-GMP) second-messenger pathway, a ubiquitous signaling method in bacteria. The crystal structures for both classes of c-di-GMP riboswitches have been solved showing two different mechanisms for c-di-GMP recognition. These riboswitches are located in the 5'-untranslated regions of genes involved in biofilm formation, cell motility, and virulence factors. Since c-di-GMP riboswitches are prevalent in bacterial species responsible for tetanus, anthrax, botulism, and cholera among others, their characterization has direct therapeutic applications. Although the structure of both classes of riboswitches has been extensively characterized, very few experiments have been performed in vivo. The Strobel lab has synthesized a series of c-di-GMP analogues to differentially study the ligand binding characteristics in both c-di-GMP riboswitches. Several of these ligands also show resistance to intracellular cleavage and are ideal for studying the behavior of c-di-GMP riboswitches in the cell. This proposal seeks to determine the functions of c-di-GMP in the cell and define the role of the c-di-GMP riboswitch on bacterial gene expression using a multidisciplinary approach spanning biochemistry, structural biology, microbiology, and genetics.