Structure and Mechanism of SAM-responsive Riboswitches

描述（由申请人提供）：已知非编码RNA在生物信息的维持和传递的几乎每一个水平上都起着至关重要的作用。这些RNA及其组装成核糖核蛋白（RNP）执行各种任务，例如维持染色体末端，X染色体失活，前体RNA的加工和修饰，以及将蛋白质靶向特定细胞位置。我的研究项目侧重于了解非编码RNA结构和功能之间的关系。在这项提案中，我们的目标是研究一类称为核糖开关的非编码RNA，在细菌mRNA的5 '-非翻译区（5'-UTR）中发现的顺式作用元件，通过其直接结合小分子代谢物的能力来调节基因表达。这些核糖核酸调控元件控制包括B在内的许多致病菌的多种基本代谢途径。anthracis，S. aureus和M.在芽孢杆菌属物种中，超过2%的基因以这种方式控制。硫代谢是核糖开关控制的细胞代谢的重要方面之一，其通过S-腺苷甲硫氨酸（SAM）与SAM应答RNA的四个不同亚类的直接相互作用来实现。为了详细了解这些SAM响应核糖开关的结构和生物化学，我们使用X射线晶体学解决了两个独立子类的结构。在这项工作的基础上，我们建议使用X射线晶体学，结合研究和化学探测的组合来解决：（1）SAM识别的结构基础是什么，（2）RNA如何有效区分SAM和产物形式S-腺苷高半胱氨酸（SAH），（3）伴随配体结合的RNA中的构象变化是什么，以及（4）这些构象变化如何用于影响基因调控。这些研究的结果将有助于拓宽我们对基于RNA的基因调控的认识，并提供对RNA的原子水平的理解，RNA是一种有前途的抗菌治疗靶点。 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B。炭疽菌、M.结核杆菌、铜绿假单胞菌和S.金黄色。我们的工作旨在从原子水平上了解这些RNA如何通过直接结合S-腺苷甲硫氨酸的能力来调节硫代谢。这些研究有助于通过基于结构的药物设计开发这些RNA作为抗菌治疗的潜在靶标。

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.