Modified Bases in tRNA-Mediated Antitermination

描述(由申请人提供)：有大量关于tRNA修饰的化学性质的信息，包括它们对翻译准确性和效率的贡献。尽管有这些数据，但关于修饰对tRNA的结构影响以及修饰对核糖体外RNA-RNA相互作用的热力学贡献知之甚少。事实上，核磁共振研究表明，未经修饰的反密码子臂可以采用构象光谱。在革兰氏阳性菌中，tRNA分子不仅将氨基酸运送到核糖体进行蛋白质合成，而且是调节氨基酰基tRNA合成酶基因转录和参与氨基酸代谢的基因的感受器。在这些基因的mRNA中，位于翻译起始点上游的前导区以密码子特有的方式与tRNA结合。这个被称为T-box的信使核糖核酸先导基因，以tRNA依赖的方式，通过转录抗终止机制调节基因的表达。不带电荷的结合tRNA防止转录终止子的形成，并允许RNA聚合酶通读，而带电荷的结合tRNA会导致转录终止。TRNA反密码子和前导RNA之间的相互作用对这一调控系统的功能至关重要，众所周知，tRNA反密码子臂上的修饰调控了核糖体上的密码子-反密码子相互作用。这项建议的目的是：(1)通过核磁共振确定碱基修饰对tRNA反密码子臂的构象和动力学影响(2)使用ITC(3)确定反密码子前导RNA结合的能量学作为修饰状态的函数(3)通过核磁共振确定反密码子臂前导RNA复合体的结构(4)使用纯化的抗终止实验来确定不同修饰的tRNA分子的抗终止效率，以及(5)使用修饰缺失的细菌菌株来表征在革兰氏阳性病原体中修饰缺失突变体的生理学。这些研究旨在确定tRNA碱基修饰对RNA-RNA识别的物理效应与tRNA分子生物活性之间的相关性。对病原体特异性修饰的透彻了解也可能为抗菌策略确定新的靶点。

DESCRIPTION (provided by applicant): There is a wealth of information available on the chemical nature of modifications in tRNA, including their contributions to translational accuracy and efficiency. Despite this body of data, very little is known about the structural effects of modifications on tRNA and the thermodynamic contributions of modifications to RNA- RNA interactions outside the ribosome. Indeed, NMR studies have shown that unmodified anticodon arms can adopt spectrum of conformations. In Gram-positive bacteria, tRNA molecules not only shuttle amino acids to the ribosome for protein synthesis, but also are the sensors through which transcription of aminoacyl tRNA synthetase genes and genes involved in amino acid metabolism are regulated. In the mRNA of these genes, a leader region upstream from the translation start site binds tRNA in a codon specific fashion. This mRNA leader, known as the T-box, regulates expression of the gene through a transcriptional antitermination mechanism in a tRNA-dependent fashion. A bound tRNA that is uncharged prevents formation of a transcriptional terminator and allows read-through by RNA polymerase whereas a bound tRNA that is charged leads to transcription termination. The interaction between the tRNA anticodon and the leader RNA is critical to the function of this regulatory system and it is well established that modifications in the anticodon arm of tRNA modulate codon-anticodon interactions on the ribosome. The aims of this proposal are: (1) to determine by NMR the conformational and dynamical effects of base modification on the anticodon arms of tRNA (2) to determine the energetics of anticodon-leader RNA association as a function of modification state using ITC (3) to determine by NMR the structures of anticodon arm-leader RNA complexes (4) to determine the antitermination efficiencies of variously modified tRNA molecules in vitro using a purified antitermination assay and in vivo using modification deficient bacterial strains and (5) to characterize the physiology of loss-of-modification mutants in a Gram-positive pathogen. These studies are designed to identify correlations between the physical effects of tRNA base modification on RNA-RNA recognition and on the biological activity of tRNA molecules. A thorough knowledge of pathogen-specific modification may also define new targets for antimicrobial strategies.