Basis of Gene Regulation by Purine-Binding mRNAs

描述（由申请人提供）：在细菌中，广泛使用的遗传调控手段是称为核糖开关的非蛋白质编码RNA元件。这些RNA序列是在mRNA的5 '-非翻译区（5'-UTR）中发现的顺式作用元件，并通过其将小分子代谢物直接结合到受体结构域的能力来调节基因表达。配体的结合指导下游调节结构域中的互斥二级结构开关的折叠，所述下游调节结构域直接与表达机器（RNA聚合酶或核糖体）相互作用。多种基本代谢途径，包括许多细菌（包括金黄色葡萄球菌、铜绿假单胞菌和结核分枝杆菌）中嘌呤、氨基酸和辅因子的生物合成;在芽孢杆菌和相关革兰氏阳性菌种中，超过4%的基因以这种方式控制。由于这些RNA已经进化为特异性结合小分子，因此它们作为设计针对病原菌的治疗剂的新靶标已经引起了极大的兴趣。 对长期的目标，发展的核糖开关如何有效地调节基因表达的分子理解，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。该提案详细列出了一系列具体目标，涉及：（1）配体受体中的结构变异如何使单个核糖开关的调节反应能够“调谐”到它所控制的基因，（2）详细描述配体结合位点内的RNA的结构可塑性，该结构可塑性允许它被不同的嘌呤类似物识别，（3）拓宽了我们对配体与受体结合后传递到下游调控结构域从而影响生物活性的机制的理解;（4）确定了以辅酶B12为效应子的核糖开关的原子分辨率结构。为了实现这些研究目标，结合结构方法，包括X射线晶体学和小角X射线散射，生物化学方法，如化学探测和转录测定，以及生物信息学。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是有前途的抗菌剂靶点。 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S。aureus，M.结核杆菌、铜绿假单胞菌和链球菌属。我们的工作旨在从原子水平上了解这些RNA如何通过直接结合细胞代谢产物的能力来调节细菌。这些研究有助于我们进一步了解如何通过基于结构的药物设计将RNA用作抗菌治疗的靶点。

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.