Structure and Mechanism of SAM-responsive Riboswitches

描述(由申请人提供)：众所周知，非编码RNA在生物信息的维护和传输的几乎每一个层面上都发挥着关键作用。这些RNA及其组装成的核糖核蛋白(RNPs)执行各种任务，如维持染色体末端，X染色体失活，前RNAs的处理和修饰，以及将蛋白质靶向特定的细胞位置。我的研究项目重点是了解非编码RNA结构和功能之间的关系。在这项提议中，我们旨在研究一类名为核糖开关的非编码RNA，这是一种发现于细菌mRNA5‘-非翻译区(5’-UTR)的顺式作用元件，通过直接结合小分子代谢物的能力来调节基因的表达。在包括炭疽杆菌、金黄色葡萄球菌和结核分枝杆菌在内的多种病原菌中，这些核调控元件控制着多种基本的代谢途径；在芽孢杆菌中，超过2%的基因以这种方式受到控制。硫代谢是核糖开关调控细胞代谢的重要方面之一，它通过S-腺苷甲硫氨酸与四种不同的腺苷蛋氨酸应答RNA亚类的直接相互作用而发挥作用。为了对这些SAM反应的核糖开关进行详细的结构和生化理解，我们使用X射线结晶学解决了两个不同亚类的结构。在这项工作的基础上，我们建议使用X射线结晶学、结合研究和化学探测相结合的方法来解决：(1)SAM识别的结构基础是什么，(2)RNA如何有效地区分SAM和产物S-同型半胱氨酸(SAH)，(3)伴随配体结合的RNA的构象变化，以及(4)这些构象变化如何用于基因调控。这些拟议的研究结果将有助于拓宽我们对基于RNA的基因调控的知识，并提供对作为抗菌治疗靶点的RNA的原子水平的理解。公共卫生相关性：核糖开关是一种基于RNA的基因调控形式，广泛用于细菌，包括许多医学上重要的病原菌，如炭疽杆菌、结核分枝杆菌、铜绿假单胞菌和金黄色葡萄球菌。我们的工作旨在从原子水平上了解这些RNA是如何通过直接结合S-腺苷蛋氨酸的能力来调节硫代谢的。这些研究有助于通过基于结构的药物设计将这些RNA开发为抗菌治疗的潜在靶点。

DESCRIPTION (provided by applicant): Non-coding RNA is known to play crucial roles at almost every level of the maintenance and transmission of biological information. These RNAs and their assemblies into ribonucleoproteins (RNPs) perform diverse tasks such as maintaining the ends of chromosomes, X-chromosome inactivation, processing and modification of pre-RNAs, and the targeting of proteins to specific cellular locations. My research program focuses on understanding the relationship between non-coding RNA structure and function. In this proposal, we aim to study a class of non-coding RNAs called riboswitches, cis-acting elements found in the 5'-untranslated region (5'-UTR) of bacterial mRNAs that regulate gene expression via their ability to directly bind small molecule metabolites. These riboregulatory elements control a variety of basic metabolic pathways in a number of pathogenic bacteria, including B. anthracis, S. aureus and M. tuberculosis; in Bacillus species, over 2% of all genes are controlled in this fashion. Sulfur metabolism is one of the most important aspects of cellular metabolism controlled by riboswitches, which is effected through direct interaction of S-adenosylmethionine (SAM) with four distinct subclasses of SAM-responsive RNAs. To develop a detailed structural and biochemical understanding of these SAM-responsive riboswitches, we have solved the structure of two separate subclasses using X-ray crystallography. Building from this work, we propose to use a combination of X-ray crystallography, binding studies and chemical probing to address: (1) what is the structural basis for SAM recognition, (2) how does RNA effectively discriminate between SAM and the product form S- adenosylhomocysteine (SAH), (3) what are the conformational changes in the RNA that accompany ligand binding, and (4) how are these conformational changes used to effect gene regulation. The results of these proposed studies will serve to broaden our knowledge of RNA-based gene regulation as well as provide an atomic-level understanding of an RNA that is a promising antimicrobial therapeutic target. PUBLIC HEALTH RELEVANCE: Riboswitches are a form of RNA-based gene regulation that is widely utilized in bacteria, including a number of medically important pathogenic bacteria such as B. anthracis, M. tuberculosis, P. aeruginosa and S. aureus. Our work seeks to develop an atomic-level understanding of how these RNAs regulate sulfur metabolism through their ability to directly bind S-adenosylmethionine. These studies serve to develop these RNAs as potential targets of antibacterial therapeutics via structure-based drug design.