Structure and Function of S-adenosyl-L-methionine Riboswitches

描述（由申请人提供）：核糖开关是识别特定小分子（通常是关键代谢物）的调节性RNA，并在转录或翻译水平上“切换”下游基因表达的开启或关闭。这些短顺式作用 RNA 元件的发现极大地改变了我们对遗传调控机制的理解。核糖开关在革兰氏阳性细菌中尤其普遍，例如作为模式生物的枯草芽孢杆菌，但也发现其控制重要病原体中的必需基因，例如炭疽芽孢杆菌、葡萄球菌、肠球菌、链球菌、李斯特菌、梭菌和分枝杆菌。这一特征和其他特征引起了人们对核糖开关介导的调节的越来越多的关注。三种不同类别的 S-腺苷甲硫氨酸 (SAM) 核糖开关是自然界中最常见的核糖开关类别。这些RNA代表了实现特定SAM识别的三种独立的进化解决方案。我们最近确定了两类 SAM 核糖开关的晶体结构：粪肠球菌 SMK 盒和枯草芽孢杆菌 S 盒。这些结构揭示了 SAM 是如何被特异性识别的，但没有提供足够的证据来支持先前遗传和生化研究中观察到的依赖于 SAM 的大构象变化。为了充分理解它们的结构-功能关系和构象动力学，我们建议：（1）了解SMK盒核糖开关中的配体识别机制。 (2) 表征无配体SMK构象并寻找真核核糖开关。 (3) 进行化学探测实验，揭示配体诱导的SMK RNA构象动力学 公共健康相关性：所描述的 SAM 核糖开关结构功能研究包括合理设计可能含有抗生素活性的特定核糖开关抑制剂的实际应用，以及为研究界开发荧光 RNA 标记系统来跟踪活细胞中 RNA 的位置、浓度和运动。

DESCRIPTION (provided by applicant): Riboswitches are regulatory RNAs that recognize specific small molecules, usually key metabolites, and "switch" downstream gene expression on or off at either the transcriptional or translational level. The discovery of these short cis-acting RNA elements has drastically changed our understanding of genetic regulatory mechanisms. Riboswitches are especially prevalent in Gram-positive bacteria, exemplified by Bacillus subtilis as a model organism, but are also found to control essential genes in important pathogens such as Bacillus anthracis, Staphylococcus, Enterococcus, Streptococcus, Listeria, Clostridium, and Mycobacterium. This and other characteristics have attracted increasing attention to riboswitch-mediated regulation. The three distinct classes of S-adenosyl methionine (SAM) riboswitches are the most commonly found riboswitch classes in nature. These RNAs represent three independent evolution solutions to achieve specific SAM recognition. We recently determined the crystal structures of SAM riboswitches from two classes, the E. faecalis SMK box and the B. subtilis S box. These structures shed light into the how SAM is specifically recognized, but did not provide enough evidence to support the large SAM-dependent conformational changes observed in the previous genetic and biochemical studies. To fully understand their structure-functional relationship and conformational dynamics, we propose to: (1) Understand the ligand recognition mechanism in the SMK box riboswitch. (2) Characterize the ligand-free SMK conformation and search for eukaryotic riboswitches. (3) Carry out chemical probing experiments to reveal ligand-induced conformational dynamics in the SMK RNA PUBLIC HEALTH RELEVANCE: The described structure-function studies of SAM riboswitches include practical applications to rationally design specific riboswitch inhibitors that may contain antibiotic activities and to develop a fluorescent RNA tagging system for the research community to track the location, concentration, and movement of RNA in living cells.