Basis of Gene Regulation by Purine-Binding mRNAs

描述(申请人提供)：在细菌中，一种广泛使用的遗传调节手段是一种称为核糖开关的非蛋白质编码RNA元件。这些RNA序列是位于mRNAs 5‘-非翻译区(5’-UTR)的顺式作用元件，它们通过直接将小分子代谢物与受体结构域结合来调节基因表达。配体的结合指导下游调控结构域中相互排斥的二级结构开关的折叠，该结构域直接与表达机制(RNA聚合酶或核糖体)对接。在许多细菌中，包括嘌呤、氨基酸和辅因子生物合成的各种基本代谢途径，包括金黄色葡萄球菌、铜绿假单胞菌和结核分枝杆菌；在芽孢杆菌和相关的革兰氏阳性物种中，超过4%的基因以这种方式控制。由于这些RNA已经进化成专门结合小分子的分子，它们作为设计针对病原体的治疗药物的新靶点已经引起了极大的兴趣。为了发展对核糖开关如何有效调节基因表达的分子理解的长期目标，嘌呤结合核糖开关将被用作解决结构和机制问题的模型系统。这项建议详细介绍了一系列具体目标，旨在解决：(1)配体受体的结构变化如何使单个核糖开关的调控反应能够对其控制的基因“调谐”，(2)详细说明配体结合位点内的RNA的结构可塑性，使其能够被不同的嘌呤类似物识别，(3)拓宽我们对配体与受体结合的机制的理解，从而影响生物活性，以及(4)确定其效应者为辅酶B12的核糖开关的原子分辨结构。为了实现这些研究目标，将结构方法(包括X射线结晶学和小角X射线散射)、生化方法(如化学探测和转录分析)以及生物信息学相结合。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，并提供对有望成为抗菌剂靶标的RNA的原子水平的理解。 与公共卫生相关：核糖开关是一种基于RNA的基因调控形式，广泛用于细菌，包括一些医学上重要的病原菌，如金黄色葡萄球菌、结核分枝杆菌、铜绿假单胞菌和链球菌。我们的工作旨在从原子层面了解这些RNA是如何通过直接结合细胞代谢物的能力来调节细菌的。这些研究有助于加深我们对RNA如何通过基于结构的药物设计作为抗菌治疗靶点的理解。

DESCRIPTION (provided by applicant): In bacteria, a widely used means of genetic regulation is a non-protein coding RNA element called a riboswitch. These RNA sequences are cis-acting elements found in the 5'-untranslated region (5'-UTR) of mRNAs and regulate gene expression via their ability to directly bind small molecule metabolites to a receptor domain. Binding of the ligand directs the folding of a mutually exclusive secondary structural switch in a downstream regulatory domain that directly interfaces with the expression machinery (either RNA polymerase or the ribosome). A variety of basic metabolic pathways including purine, amino acid, and cofactor biosynthesis in a number of bacteria, including Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis; in Bacillus and related Gram-positive species, over 4% of all genes are controlled in this fashion. Since these RNAs have already evolved to specifically bind small molecules, they have become of great interest as novel targets for designing therapeutics targeted against pathogenic bacteria. Towards the long-term goal of developing a molecular understanding of how riboswitches efficiently regulate gene expression, purine-binding riboswitches will be employed as a model system for addressing structural and mechanistic questions. This proposal details a set of specific aims that address: (1) how structural variation in the ligand receptor enables the regulatory response of individual riboswitches to be "tuned" to the gene it controls, (2) detail the structural plasticity of the RNA within the ligand binding site that allows it to be recognized by diverse purine analogs, (3) broaden our understanding into the mechanism by which ligand binding to the receptor is communicated to downstream regulatory domain to effect biological activity, and (4) determine the atomic-resolution structure of a riboswitch whose effector is coenzyme B12. To address these research goals, a combination of structural approaches including X-ray crystallography and small angle X-ray scattering, biochemical approaches such as chemical probing and transcriptional assays, and bioinformatics. 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 RNAs that are promising targets of antimicrobial agents. 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 S. aureus, M. tuberculosis, P. aeruginosa and Streptococcus species. Our work seeks to develop an atomic-level understanding of how these RNAs regulate bacterial through their ability to directly bind cellular metabolites. These studies serve to further our understanding into how RNA can be exploited as targets of antibacterial therapeutics via structure-based drug design.