Dynamic basis of the molecular mechanism of nucleotide secondary messengers-sensing riboswitches by NMR spectroscopy

环二核苷酸如c-di-GMP、c-di-AMP和c-AMP-GMP以及ZTP是调节多种不同基因表达的核苷酸二级信使，这些基因涉及细菌的生物膜形成、运动性、细胞壁生物合成、毒力和外生电作用。基因表达的调控是基于这些核苷酸二级信使与核糖开关的结合。核糖开关是mRNA5-UTRs中基于RNA的调节元件，能够调节转录或翻译。在这个项目中，我们将研究核糖开关介导的基因调控的分子基础。特别是，局部以及大范围的构象动力学是核糖开关功能的核心。我们将通过应用先进的溶液多维异核核磁共振波谱来研究环二核苷酸和ZTP-核糖开关的功能动力学，从而在原子分辨率下研究这些动态过程。特别是，我们将重点阐明配体结合引起的结构变化，适体结构域水平上核糖开关的功能动力学，全长核糖开关以及重要的转录中间产物。按照这种方法，我们将建立适配子结构域和表达平台之间构象交互作用的动态框架的分子基础，以及它们显著的配体选择性的分子基础。我们的项目将有助于更好地了解这些核糖开关的功能的分子基础，将揭示使类似的分子功能适应不同环境条件的分子策略，并可能有助于设计具有新底物特性的核糖开关，例如用于合成生物学方法中的人工配体类似物。

The cyclic dinucleotides such as c-di-GMP, c-di-AMP and c-AMP-GMP as well as ZTP are nucleotide secondary messengers that regulate the expression of a multitude of different genes involved for instance in biofilm formation, motility, cell wall biosynthesis, virulence and exoelectrogenesis in bacteria. Regulation of gene expression is based on binding of these nucleotide secondary messengers to riboswitches. Riboswitches are RNA-based regulatory elements in the 5-UTRs of mRNAs capable of regulating transcription or translation. In this project, we will investigate the molecular basis of the riboswitch-mediated gene regulation. In particular, local as well as large scale conformational dynamics are at the heart of riboswitch function. We will investigate these dynamic processes at atomic resolution by applying advanced solution multidimensional heteronuclear NMR-spectroscopy to investigate the functional dynamics of the cyclic dinucleotide and the ZTP-riboswitches. In particular, we will on focus on elucidating the structural changes induced by ligand binding, the functional dynamics of the riboswitches on the level of the aptamer domains, the full-length riboswitches as well as functionally important transcriptional intermediates. Following this approach, we will establish the molecular basis for the dynamic framework of the conformational cross-talk between the aptamer domain and the expression platforms which underlies riboswitch function as well as the molecular basis for their remarkable ligand selectivity. Our project will contribute to a better understanding of the molecular basis for the function of these riboswitches, will uncover molecular strategies to adapt similar molecular functionalities to different environmental conditions and might be helpful to enable the design of riboswitches with novel substrate specifities e. g. for artificial ligand analogues in synthetic biology approaches.