Single molecule FRET study of viral programmed ribosomal frameshifting

病毒程序性核糖体移码的单分子 FRET 研究

• 批准号: 8784140
• 负责人: Ryan Michael Jamiolkowski
• 金额: $ 4.68万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8784140
• 关键词: Base Sequence; Behavior; Biochemistry; Cells; Codon Nucleotides; Data; Doctor of Philosophy; Energy Transfer; Fellowship; Fluorescence Resonance Energy Transfer; Goals; Guanosine Triphosphate Phosphohydrolases; HIV; Individual; Infectious bronchitis virus; Instruction; Kinetics; Label; Measurement; Measures; Messenger RNA; Modeling; Molecular Conformation; Nucleotides; Peptide Elongation Factor G; Physicians; Physiological; Process; Production; Program Efficiency; Property; Proteins; Reading Frames; Research; Research Training; Resolution; Ribosomal Frameshifting; Ribosomes; Scientist; Severe Acute Respiratory Syndrome; Site; Specific qualifier value; Structure; Students; System; Techniques; Testing; Time; Translating; Translations; Viral; Viral Genome; Viral Pathogenesis; Virion; Virulence; Virus; West Nile virus; Work; Yeasts; cofactor; design; fluorophore; insight; macromolecule; novel; particle; pathogen; programs; public health relevance; reproductive; research study; single molecule; single-molecule FRET; stem; stoichiometry; therapeutic target; virology

DESCRIPTION (provided by applicant): This application is for an individual fellowship for an MD-PhD student, with a research training plan designed to aid his long-term goal of becoming an independent physician scientist in the field of virology. Many viruses (including HIV, SARS, and West Nile) evolved an ability to regularly trigger frameshifts by one nucleotide~ these viral programmed ribosomal frameshifts (PRFs) produce proteins from overlapping reading frames of the same mRNA. It is unknown what structural changes occur in the ribosome during PRF, or how downstream mRNA 2¿ structures enhance the efficiency of PRF at 7-nucleotide "slippery sequences." This application proposes using single molecule FRET (smFRET) to study dynamic, frameshifting ribosomes to distinguish between competing models of PRF mechanism. Preliminary smFRET characterization of the behavior of dynamically translating ribosomes when not undergoing PRF revealed a novel state seen only during active translation. In aim 1, smFRET study of dynamic prokaryotic and eukaryotic ribosomes will be extended to determine if that novel conformation corresponds to global structural changes during multiple substeps of translation. In aim 2, smFRET of dynamic ribosomes on viral mRNA will be used to determine the mechanism of PRF, namely the structural and kinetic properties unique to ribosomes that frameshift, how mRNA 2¿ structures enhance PRF efficiency, and mechanistic similarities and differences of PRF in multiple viruses (IBV, SARS, HIV). These experimental results will distinguish between competing models of PRF, giving a detailed understanding of this process central to viral pathogenesis.

描述(由申请人提供)：此申请是为一名医学博士学生提供的个人奖学金，其研究培训计划旨在帮助他成为病毒学领域的一名独立内科科学家的长期目标。许多病毒(包括HIV、SARS和西尼罗河病毒)进化出一种通过一个核苷酸定期触发移码的能力。这些病毒编程的核糖体移码(PRF)从相同mRNA的重叠阅读框架中产生蛋白质。目前尚不清楚在PRF过程中核糖体发生了什么结构变化，也不知道下游的mRNA2？结构如何在7个核苷酸的“光滑序列”上提高PRF的效率。本申请建议使用单分子FRET(SmFRET)来研究动态的移码核糖体，以区分PRF机制的竞争模型。对没有经历PRF时动态翻译核糖体的行为的初步smFRET表征揭示了一种只有在主动翻译过程中才能看到的新状态。在目标1中，将扩大对动态原核和真核核糖体的smFRET研究，以确定该新构象是否对应于翻译的多个子步骤中的整体结构变化。在目标2中，将利用动态核糖体对病毒mRNA的smFRET来确定PRF的机制，即移码的核糖体所特有的结构和动力学性质，mRNA2‘结构如何提高PRF的效率，以及多种病毒(IBV，SARS，HIV)中PRF机制的异同。这些实验结果将区分不同的PRF竞争模型，从而详细了解这一病毒致病机制的核心过程。