Single molecule translational profiling

描述(申请人提供)：翻译是基因表达的终点。信使RNA是使用一种以核糖体为核心的复杂机器进行解码的。尽管它很重要，但追踪细胞mRNAs翻译的方法仍然没有开发出来。在此，我们基于多年来单分子翻译方法的发展，提出了一种新颖的、实时的密码子分辨率的翻译跟踪方法。我们的方法使用荧光标记的tRNA、核糖体和其他配体来定位翻译起点、编码序列和终止位点。我们在初步数据的基础上证明，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。我们将在拟议的资助期内将我们的开发努力集中在三个具体目标上，这三个目标将(1)创建一个用于单分子翻译图谱的基准荧光试剂集合(2)使用这些试剂来表征三种生物：大肠杆菌、酵母和人类的翻译起点、阅读框架、终止位点和药物效应，以及(3)表征这些生物中罕见的翻译现象，如移码。这项工作的目标是对这些生物体的翻译过程提供实时的、全基因组的分析。这项提议的终点将是一个核心研究设施，致力于单分子翻译分析，并提供进入更广泛的生物医学界的途径。我们相信，这些结果将使我们更深入地理解翻译调控在人类疾病中的作用。

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.