Single molecule translational profiling

单分子翻译分析

• 批准号: 8181683
• 负责人: JOSEPH D PUGLISI
• 金额: $ 147.2万
• 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/8181683
• 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. PUBLIC HEALTH RELEVANCE: Translation is a central process of gene regulation. Here, we propose a radically novel new approach to translational profiling using single-molecule fluorescence detection in real time.

描述（由申请人提供）：翻译是基因表达的终点。信使 RNA 使用以核糖体为核心的复杂机器进行解码。尽管其重要性，追踪细胞 mRNA 翻译的方法仍未开发出来。在这里，我们以多年单分子翻译方法开发为基础，提出了一种新颖的实时方法来跟踪密码子分辨率的翻译。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的图谱。我们的初步数据表明，利用最近开发的 DNA 测序仪器，可以使用荧光标记的 tRNA 和核糖体进行实时单分子翻译分析。在拟议的资助期内，我们将把开发工作重点放在三个具体目标上，即（1）创建一系列用于单分子翻译分析的基准荧光试剂（2）使用这些试剂来表征翻译起始位点、阅读框、终止位点以及药物在三种生物体中的作用：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，例如移码。这项工作的目标是对这些生物体的翻译过程进行实时、全基因组分析。该提案的终点将是一个核心研究设施，致力于单分子转化分析并为更广泛的生物医学界提供机会。我们相信这些结果将有助于更深入地了解翻译调控在人类疾病中的作用。 公共卫生相关性：翻译是基因调控的核心过程。在这里，我们提出了一种利用实时单分子荧光检测进行翻译分析的全新方法。