Investigation of Ligand Binding and Gene Regulation by the GEMM Riboswitch

GEMM 核糖开关的配体结合和基因调控研究

• 批准号: 7676916
• 负责人: ALISON LIVINGSTON
• 金额: $ 4.81万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7676916
• 关键词: Adopted; Affect; Affinity; Antibiotics; Archaea; Bacteria; Base Pairing; Binding; Biochemical; Cell physiology; Cells; Characteristics; Complement; Development; Drug Delivery Systems; Electron Transport; Electrophoretic Mobility Shift Assay; Elements; Employment; Environment; Eukaryota; Feedback; Fourier Analysis; Gene Expression; Gene Expression Regulation; Genes; Goals; Hybrids; Indium; Investigation; Ligand Binding; Ligands; Membrane; Messenger RNA; Modification; Molecular; Monitor; Mutagenesis; Mutation; Organism; Pathogenesis; Phenotype; Process; RNA; RNA Folding; Second Messenger Systems; Signal Transduction; Specificity; Structure; Techniques; Testing; Time; Untranslated Regions; Virulence; Work; aptamer; base; cell motility; extracellular; in vivo; insight; interest; novel; pathogenic bacteria; plant fungi; public health relevance; recombinant RNA; research study; response; second messenger; sensor; small molecule; stem

DESCRIPTION (provided by applicant): Riboswitches are RNA sensors located in untranslated regions of mRNA that regulate expression of adjacent genes in bacteria and some eukaryotes. In many cases, riboswitches act through a feedback mechanism by binding target small molecule ligands that are critical elements in important cellular processes. A recently discovered class of riboswitches, termed GEMM, was found upstream of genes that influence membranes, motility, or the extracellular environment. Predicted secondary structural elements of the RNA include two highly conserved stems whose internal loops are thought to confer ligand binding specificity. Experiments have demonstrated that cyclic diguanosine monophosphate (c-diGMP), an important second messenger molecule, binds to the GEMM riboswitch with high affinity and selectivity. Cellular functions such as motility and pathogenesis were previously associated with fluctuations in c-diGMP concentration, although the molecular basis for this observation is presently unknown. The goal of this work is to explore the basis of c-diGMP ligand binding and gene regulation by the GEMM riboswitch using both biochemical and structural analysis. The specific contacts to the RNA molecule required-for binding of c-diGMP will be established by mutagenesis (aim 1) and the dynamics of ligand binding probed by real-time NMR (aim 2). This work will complement current efforts toward an x-ray crystal structure and will help to reveal structural changes undertaken by the RNA molecule upon ligand binding. Modulation of gene expression by c-diGMP in both wild-type and hybrid GEMM riboswitch structures in a cellular context will be tested by monitoring the electron transfer ability of the bacteria Geobacteria sulfurreducens (aim 3). PUBLIC HEALTH RELEVANCE: GEMM riboswitches are gene control elements found to influence gene expression in a variety of organisms, including several pathogenic bacteria. These elements are responsive to fluctuations in the second messenger molecule cyclic diGMP and act to modulate expression of genes important for cell motility and virulence, among others. Understanding the molecular basis by which gene control is accomplished, through both structural and biochemical analysis, may help to establish the GEMM riboswitch as a potential drug target.

描述(申请人提供)：核糖开关是位于mRNA的非翻译区的RNA传感器，调节细菌和一些真核生物中相邻基因的表达。在许多情况下，核糖开关通过与目标小分子配体结合来发挥反馈作用，小分子配体是重要细胞过程中的关键元素。最近发现了一类核糖开关，称为GEMM，它位于影响细胞膜、运动性或细胞外环境的基因的上游。预测的RNA二级结构元件包括两个高度保守的茎，其内部环被认为具有配体结合的特异性。实验证明，作为重要的第二信使分子，环二鸟苷一磷酸(c-diGMP)与GEMM核糖开关具有很高的亲和力和选择性。细胞功能，如运动性和致病机制，以前与c-diGMP浓度的波动有关，尽管这种观察的分子基础目前尚不清楚。本工作的目的是通过生化和结构分析来探索GEMM核糖开关与c-diGMP配体结合和基因调控的基础。C-diGMP结合所需的与RNA分子的特定接触将通过突变(目标1)和实时核磁共振探测配体结合的动力学(目标2)来建立。这项工作将补充目前对X射线晶体结构的努力，并将有助于揭示RNA分子在配体结合时发生的结构变化。C-diGMP对野生型和杂交GEMM核糖开关结构中基因表达的调节将通过监测细菌硫磺还原地杆菌的电子转移能力(目标3)来测试。与公共卫生相关：GEMM核糖开关是一种基因控制元件，可影响多种生物体的基因表达，包括几种致病菌。这些元件对第二信使分子环状diGMP的波动做出反应，并调节对细胞运动和毒力至关重要的基因的表达等。通过结构和生化分析了解基因控制的分子基础，可能有助于将GEMM核糖开关确立为潜在的药物靶点。