Polysomes & Subunits: Structure-Function Relationships

描述(由申请者提供)：摘要：这项资助计划的长期目标是在原子水平上了解核糖体在蛋白质合成过程中的功能。我们将采取多学科的方法，利用遗传学、生化和结构方法来研究细菌核糖体。我们的第一个目标是研究核糖体结构在mRNA解码过程中的动态以及抗生素对其的抑制作用。我们将使用X射线结晶学来获得突变的Thermus thermophilus 30S亚基的高分辨率结构，这些结构揭示了新的构象状态。我们将研究突变对解码的准确性和动力学的影响。我们的第二个目标是探索一条途径，通过核糖体蛋白S12和氨基酰tRNA将密码子识别信号传递到EF-Tu，以激活GTP水解，这是tRNA选择过程中的关键步骤。这些实验旨在提供对核糖体结构和功能的基本见解，包括核糖体的新构象状态，以帮助理解解码过程。解决具有抗生素耐药突变的核糖体结构的能力将有助于了解抗生素的作用和耐药机制，并对合理设计抗耐药微生物病原体的药物具有特殊的临床意义。 公共卫生相关性：项目叙述：该项目的目标是利用结构生物学、遗传学和生物化学的方法，在原子水平上加深我们对蛋白质合成的分子机制的理解。细菌中的蛋白质合成是所有抗生素的靶标，而这一过程中涉及的成分的修改和突变可能会导致抗生素耐药性，这是我们这个时代最紧迫的医学问题之一。破译抗生素耐药性的分子基础将为合理设计有效对抗抗生素耐药性微生物的新型抗菌剂提供重要信息。对蛋白质合成的结构基础的更全面的研究也将为所有生命形式中发现的最基本的过程之一提供重要的见解。

DESCRIPTION (provided by applicant): ABSTRACT: The long-term goal of this grant proposal is to understand at an atomic level how the ribosome functions during protein synthesis. We will take a multidisciplinary approach utilizing genetic, biochemical and structural methods to study the bacterial ribosome. Our first aim is to examine ribosome structural dynamics during decoding of mRNA and its inhibition by antibiotics. We will use X-ray crystallography to obtain high-resolution structures of mutant Thermus thermophilus 30S subunits that reveal new conformational states. We will examine the effects of the mutations on the accuracy and kinetics of decoding. Our second aim is to probe a pathway by which codon recognition is signaled via ribosomal protein S12 and aminoacyl-tRNA to EF-Tu to activate GTP hydrolysis, a key step in the tRNA selection process. These experiments are designed to provide fundamental insights into ribosome structure and function, including new conformational states of the ribosome, to aid in understanding the decoding process. The ability to solve structures of ribosomes with antibiotic-resistance mutations will aid in understanding the mechanisms of antibiotic action and resistance, and has particular clinical relevance for the rational design of drugs to fight resistant microbial pathogens. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE: The goal of this project is to further our understanding of the molecular mechanisms of protein synthesis at the atomic level, using the methods of structural biology, genetics and biochemistry. Protein synthesis in bacteria is the target of over half of all antibiotics, and modifications and mutations in components involved of this process can lead to antibiotic resistance, one of the most pressing medical problems of our time. Deciphering the molecular basis for antibiotic resistance will provide important information needed for the rational design of novel antimicrobial agents effective against antibiotic resistant microorganisms. A more comprehensive examination of the structural basis for protein synthesis will also provide important insights into one of the most fundamental processes found in all forms of life.