Modified Bases in tRNA-Mediated Antitermination

tRNA 介导的抗终止中的修饰碱基

基本信息

批准号：
7725576
负责人：
EDWARD P NIKONOWICZ
金额：
$ 1.78万
依托单位：
RICE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-01-15 至 2010-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7725576
关键词：
Adopted Amino Acids Amino Acyl-tRNA Synthetases Anticodon Binding Binding Sites Biological Biological Assay Boxing Calorimetry Charge Chemicals Codon Nucleotides Collaborations Complex DNA-Directed RNA Polymerase Data Elements Environment Exhibits Gene Expression Genes Genetic Transcription Goals Gram-Positive Bacteria Heteronuclear NMR In Vitro Individual Knowledge Letters Ligands Measures Mediating Messenger RNA Metabolism Modification Molecular Molecular Conformation NMR Spectroscopy Nature Nucleotides Ohio Physiological Physiology Play Process Property Protein Biosynthesis RNA Reading Research Design Research Personnel Ribosomes Role Solutions Specific qualifier value Structure System Testing Thermodynamics Titrations Transcriptional Regulation Transfer RNA Translations Triplet Multiple Birth Universities Upper arm Work antimicrobial antitermination base chemical property in vivo medical schools mutant pathogen prevent programs research study sensor stem transcription termination

项目摘要

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.

描述（由申请人提供）：有大量有关tRNA修改化学性质的信息，包括它们对翻译准确性和效率的贡献。尽管有这些数据，但对修饰对tRNA的结构效应以及对核糖体以外RNA-RNA相互作用的热力学贡献的结构效应知之甚少。实际上，NMR的研究表明，未修改的反密码子可以采用大量构象。在革兰氏阳性细菌中，tRNA分子不仅将氨基酸穿梭至核糖体进行蛋白质合成，而且是调节氨基酰基TRNA合成酶基因的转录以及与氨基酸代谢相关的基因的转录的传感器。在这些基因的mRNA中，翻译起始位点上游的领导区域以特定于密码子的方式结合tRNA。这个被称为T-box的mRNA领导者以tRNA依赖性方式通过转录抗验证机制调节基因的表达。未充电的结合tRNA阻止了转录终止剂的形成，并允许通过RNA聚合酶读取，而充电的结合tRNA会导致转录终止。 tRNA反密码子与Leader RNA之间的相互作用对于该调节系统的功能至关重要，并且可以很好地确定，tRNA的反密码子中的修饰调节核糖体上的密码子 - 抗原相互作用。该提案的目的是：（1）通过NMR确定基础修饰对tRNA（2）的反登基臂的构象和动力学效应，以确定使用ITC（3）的反激长RNA关联的能量学，以通过NMR来确定摩托子臂rna复合物的结构（4）的变化状态（3）使用纯化的抗授权测定法和体内使用修饰缺陷细菌菌株和（5）在体外进行体外表征，以表征革兰氏阳性病原体中修复丧失突变体的生理学。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分子的生物学活性之间的相关性。对病原体特异性修饰的透彻知识也可以定义抗菌策略的新目标。