Structural Requirements for Ribosome-Dependent GTPase Activation and Hybrid State

核糖体依赖性 GTP 酶激活和混合状态的结构要求

• 批准号: 8626683
• 负责人: Paul Clinton Spiegel
• 金额: $ 28.41万
• 依托单位: WESTERN WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8626683
• 关键词: Address; Adopted; Antibiotic Resistance; Antibiotics; Bacterial Infections; Behavior; Binding; Biochemistry; Biological; Biological Assay; Budgets; Catalysis; Cells; Chemicals; Clinical; Clinical Treatment; Collaborations; Commit; Complex; Development; Drug Targeting; Educational process of instructing; Elongation Factor; Equilibrium; Fluorescence Resonance Energy Transfer; Funding; Guanine Nucleotides; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hereditary Disease; Hybrids; Lead; Letters; Life; Malignant Neoplasms; Measurement; Measures; Messenger RNA; Methods; Molecular; Molecular Conformation; Molecular Machines; Movement; Mutation; Nature; Nucleotides; Peptide Elongation Factor G; Pharmaceutical Preparations; Phase; Platelet Factor 4; Preparation; Procedures; Process; Protein Biosynthesis; Public Health; RNA Binding; Recombinants; Research; Research Project Grants; Ribosomal Proteins; Ribosomes; Roentgen Rays; Role; Rotation; Solutions; Structure; Techniques; Time; Toxic effect; Transfer RNA; Translations; Universities; Work; conformer; deafness; graduate student; insight; macromolecule; pathogenic bacteria; prevent; public health relevance; reconstitution; single-molecule FRET; translocase; undergraduate research

Summary ! During protein synthesis, transfer RNAs (tRNAs) and messenger RNA (mRNA) move in a coordinated manner through the ribosome, a process denoted as 'ribosomal translocation'. As translocation occurs, many transient intermediates are formed whereby ribosome-bound tRNA substrates adopt several different conformations. Concomitantly, the 70S ribosome rotates through different conformers to accommodate the movement of tRNA during the elongation phase of translation. This process is strictly regulated by the ribosome-dependent GTPase elongation factor G (EF-G), which binds transiently to the ribosome subunit interface where GTP is hydrolyzed to allow for rapid and accurate translation to proceed. In this study, chemical footprinting and intersubunit FRET procedures will be employed to study the structural requirements of EF-G for stabilization of the first step of ribosomal translocation, the formation of the hybrid state. Secondly, the nature of the interaction between a newly described ribosomal translocase, termed elongation factor 4 (EF4), and the 70S ribosome under different translocation states will be further described. Third, the role of ribosomal protein L7/L12 in ribosome-dependent GTPase binding and activation by the 70S ribosome will be defined through GTPase activity assays and methods for the quantitative measure of EF-G equilibrium binding. Lastly, the dynamic, solution structure states of several ribosome-dependent GTPases in the presence of different guanine nucleotides will be characterized with small angle x-ray scattering (SAXS) approaches to study biological macromolecules. In this study, we propose to investigate for the first time the domain requirements of EF-G for stabilization of the hybrid state and the structural mechanism of EF4-catalyzed reverse translocation in solution. Furthermore, we hypothesize that the highly purified preparations of ribosomes depleted in ribosomal protein L7/L12, as well as a recombinant form of L7/L12 for reconstitution, will lead to an unambiguous description of its role in GTPase binding and catalysis. Lastly, we will demystify the dynamic nature of ribosome-dependent GTPases in regards to the recognition of ribosome functional complexes. This proposed work will capitalize on the PI's expertise in footprinting, SAXS, and ribosome biochemistry. Public Health Relevance: The most effective antibiotics used for clinical treatments of bacterial infections target ribosomes. Therefore, a better understanding of the structure and function of the ribosome will aid in finding new drugs that address the increasing problem of antibiotic-resistant pathogenic bacteria. Furthermore, mutations that occur within ribosomes that prevent proper function have been associated with genetic diseases, such as deafness and cancer. !

总结