Investigation of Ligand Binding and Gene Regulation by the GEMM Riboswitch

描述（由申请人提供）：核糖开关是位于mRNA非翻译区域的RNA传感器，可调节细菌和一些真核生物中邻近基因的表达。在许多情况下，核开关通过反馈机制结合靶小分子配体，这些配体是重要细胞过程中的关键元素。最近发现的一类核开关，称为GEMM，是在影响膜、运动或细胞外环境的基因上游发现的。预测的RNA二级结构元件包括两个高度保守的茎，其内部环被认为赋予配体结合特异性。实验表明，环二瓜苷单磷酸（c-diGMP）是一种重要的第二信使分子，具有高亲和力和选择性地与GEMM核糖开关结合。细胞功能，如运动和发病机制，以前与c-diGMP浓度的波动有关，尽管这一观察的分子基础目前尚不清楚。这项工作的目的是利用生化和结构分析来探索c-diGMP配体结合和GEMM核开关基因调控的基础。与c-diGMP结合所需的RNA分子的特定接触将通过诱变（目的1）和实时核磁共振探测的配体结合动力学（目的2）来建立。这项工作将补充目前对x射线晶体结构的研究，并将有助于揭示RNA分子在配体结合时所发生的结构变化。通过监测硫还原Geobacteria sulreducens细菌的电子转移能力，将测试c-diGMP在细胞背景下野生型和杂交GEMM核糖开关结构中的基因表达调节（目的3）。公共卫生相关性：GEMM核开关是一种基因控制元件，被发现影响多种生物体的基因表达，包括几种致病菌。这些元件响应第二信使分子环diGMP的波动，并调节对细胞运动和毒力等重要基因的表达。通过结构和生化分析，了解基因控制完成的分子基础，可能有助于确定GEMM核糖开关作为潜在的药物靶点。

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.