Single molecule translational profiling

单分子翻译分析

• 批准号: 8727064
• 负责人: JOSEPH D PUGLISI
• 金额: $ 72.2万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8727064
• 关键词: Bacteria; Bacterial RNA; Benchmarking; Biological Assay; Code; Codon Nucleotides; Collection; Communities; Complementary DNA; Complex; DNA Sequence; Data; Detection; Development; Development Plans; Elongation Factor; Escherichia coli; Fluorescence; Fluorescent Dyes; Funding; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genomics; Goals; Human; Internal Ribosome Entry Site; Label; Lead; Ligands; Maps; Messenger RNA; Methods; Organism; Peptide Initiation Factors; Pharmaceutical Preparations; Process; Proteins; RNA; RNA Sequences; Reading Frames; Reagent; Regulation; Resolution; Ribosomes; Role; Scheme; Signal Transduction; Site; Structure; System; Technology; Testing; Time; Transfer RNA; Translating; Translational Regulation; Translations; Viral; Work; Yeasts; arm; genome-wide; genome-wide analysis; human disease; instrumentation; method development; new technology; novel; novel strategies; research facility; research study; single molecule; termination factor; translational approach; trend

DESCRIPTION (provided by applicant): Translation is the endpoint of gene expression. Messenger RNAs are decoded using a complex machinery that has the ribosome as its centerpiece. Despite its importance, methods to track translation of cellular mRNAs remain undeveloped. Here, we build on years of method development in single-molecule translation to propose a novel, real-time method to track translation at codon resolution. Our approach uses fluorescently labeled tRNAs, ribosomes and other ligands to map translation start sites, coding sequences and termination sites. We build on our preliminary data demonstrating that real time single-molecule analysis of translation can be performed using fluorescently labeled tRNAs and ribosomes, harnessing recently-developed DNA sequencing instrumentation. We will focus our development efforts during the proposed funding period on three specific aims that will (1) create a collection of benchmarked fluorescent reagents for single-molecule translational profiling (2) use these reagents to characterize translational start sites, reading frames, termination sites and drug effects in three organisms: E. coli, yeast and human and (3) characterize rare translational phenomena, such as frameshifting, in these organisms. The goal of this work is provide real-time, genome-wide analysis of translational processes in these organisms. The endpoint of this proposal will be a core research facility that is dedicated to single-molecule translational analysis and providing access to the broader biomedical community. We believe these results will lead to a deeper understanding of the role of translational regulation in human disease.

描述（申请人提供）：翻译是基因表达的终点。信使RNA是通过一种以核糖体为核心的复杂机制来解码的。尽管其重要性，跟踪细胞mRNA翻译的方法仍然没有开发。在这里，我们建立在单分子翻译多年的方法发展，提出了一种新的，实时的方法来跟踪翻译密码子分辨率。我们的方法使用荧光标记的tRNA、核糖体和其他配体来绘制翻译起始位点、编码序列和终止位点。我们建立在我们的初步数据表明，真实的时间翻译的单分子分析可以使用荧光标记的tRNA和核糖体，利用最近开发的DNA测序仪器。在拟议的资助期内，我们将把开发工作集中在三个具体目标上，即（1）创建一系列用于单分子翻译谱分析的基准荧光试剂（2）使用这些试剂表征三种生物体中的翻译起始位点、阅读框架、终止位点和药物效应：E.大肠杆菌、酵母和人类;（3）表征这些生物体中罕见的翻译现象，如移码。这项工作的目标是提供这些生物体中翻译过程的实时，全基因组分析。该提案的终点将是一个核心研究设施，致力于单分子翻译分析，并提供更广泛的生物医学界。我们相信这些结果将导致更深入地了解翻译调控在人类疾病中的作用。