Resolving Structure and Mechanism of tRNA-actuated riboswitches

 描述(申请人提供)：摘要在过去的十年里，RNA介导的调控已经成为基因表达的一个中心主题。革兰氏阳性菌的一种常见控制机制是核糖开关。在核糖开关中，传感器结构域机械耦合到由相互排斥的终止子/反终止子RNA结构元件组成的调节域，这些结构元件可以减弱或增加翻译/转录。核糖开关的一种特别广泛的类型是“T盒”，这是一种tRNA激活的开关，其默认状态是减弱编码氨基酰-tRNA合成酶、氨基酸生物合成酶和氨基酸运输机械的必要基因的转录。高比例的带电和不带电的tRNA使开关保持在关闭状态，并抑制基因表达。核糖开关的功能受到多种因素的调节，包括保守结构元件周围的核苷酸序列变化和tRNA碱基修饰，并且对细胞的适合性至关重要。在这个建议中，我们将(1)使用SAXS和溶液核磁共振方法确定模型tRNA-核糖开关复合体的全局构象和结构细节；(2)利用单分子FRET技术建立包括tRNA修饰和氨基酰化状态等变量的T盒核糖开关机制的动力学模型；以及(3)通过直接RNA测量分析在体内确定tRNA修饰和自然序列变化对T盒调控效率的影响。也许对于任何核糖开关，我们将第一次建立一个核糖开关功能的机制框架，该框架将局部序列依赖的贡献与全局动力学和T盒效率定量地联系起来。此外，该项目还将拓宽我们对tRNA核苷酸修饰对革兰氏阳性菌基因调控和细胞适合性的贡献的认识。最后，T盒核糖开关提供了一个独特的平台，可以在其上设计基因工具来询问细菌细胞的新陈代谢状态。

 DESCRIPTION (provided by applicant): Abstract Over the past decade, RNA-mediated regulation has emerged as a central theme in gene expression. A common control mechanism of Gram-positive bacteria is the riboswitch. In riboswitches, a sensor domain is mechanistically coupled to a regulatory domain composed of mutually exclusive terminator/anti-terminator RNA structure elements that either attenuate or increase translation/transcription. A particularly widespread type of riboswitch is the "T box", a tRNA-actuated switch whose default state is to attenuate transcription of essential genes encoding aminoacyl-tRNA synthetases, amino acid biosynthetic enzymes, and amino acid transport machinery. High ratios of charged-to-uncharged tRNA maintain the switch in the off state and suppress gene expression. The performance of the riboswitch is subject to modulation by multiple factors, including nucleotide sequence variations around conserved structure elements and tRNA base modification, and is crucial to the fitness of the cell. In this proposal, we will (1) identify the global conformationsand structural details of model tRNA-riboswitch complexes using SAXS and solution NMR methods; (2) develop a kinetic model of the T box riboswitch mechanism that includes variables such as tRNA modification and aminoacylation state using single molecule FRET techniques; and (3) determine in vivo the effects of tRNA modification and natural sequence variation on T box regulatory efficiency using direct RNA measurement assays. For the first time perhaps for any riboswitch, we will establish a mechanistic framework for riboswitch function that quantitatively connects local sequence-dependent contributions with global dynamics and T box efficiency. In addition, this project will broaden our knowledge of the contributions of tRNA nucleotide modification to gene regulation and cell fitness of Gram-positive bacteria. Finally, the T box riboswitch offers a unique platform on which to engineer genetic tools to interrogate the metabolic state of bacterial cells.