Modified Bases in tRNA-Mediated Antitermination

tRNA 介导的抗终止中的修饰碱基

• 批准号: 7568953
• 负责人: EDWARD P NIKONOWICZ
• 金额: $ 30.24万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 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/7568953
• 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

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。尽管有大量数据，但人们对 tRNA 修饰的结构效应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一系列构象。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，而且还是调节氨酰tRNA合成酶基因和参与氨基酸代谢的基因转录的传感器。在这些基因的 mRNA 中，翻译起始位点上游的前导区域以密码子特异性方式结合 tRNA。这种 mRNA 前导序列被称为 T 盒，通过转录抗终止机制以 tRNA 依赖性方式调节基因的表达。不带电荷的结合 tRNA 可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 之间的相互作用对于该调节系统的功能至关重要，并且众所周知，tRNA 反密码子臂的修饰可调节核糖体上的密码子-反密码子相互作用。该提案的目的是：(1) 通过 NMR 确定 tRNA 反密码子臂上碱基修饰的构象和动力学效应 (2) 使用 ITC 确定反密码子-前导 RNA 关联的能量作为修饰状态的函数 (3) 通过 NMR 确定反密码子臂-前导 RNA 复合物的结构 (4) 确定各种修饰的 tRNA 分子的抗终止效率 在体外使用纯化的抗终止试验，在体内使用修饰缺陷的细菌菌株，以及（5）表征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 识别的物理影响与 tRNA 分子的生物活性之间的相关性。对病原体特异性修饰的全面了解也可以定义抗菌策略的新目标。