Single-molecule analysis of initial transcription in vitro and in silico

这项拟议的工作包括开发超灵敏显微镜的特殊方法，以及从理论上理解基因表达中涉及的重要遗传过程。基因表达是从遗传信息(存储在DNA中)到蛋白质制造(构成活细胞的大部分机器和结构的分子)的路径。具体地说，这项工作的重点是基因转录过程，这是由称为RNA聚合酶的蛋白质机器执行的。这些微小的生物机器读取DNA，并将信息复制到信使分子(信使RNA)中。这项拟议工作的一个特点是使用了一种特殊的显微镜(单分子荧光显微镜)。该显微镜经过精心设计，能够检测和监控检测区中存在的单个(‘单一’)荧光分子(与要求检测区中存在数千或数百万个分子的传统显微镜不同)。单分子荧光显微镜允许人们确定组成生物机器的部件的数量，测量这些部件相互结合的强度，部件之间的距离和方向，以及当微小的生物机器工作时部件的运动情况。我们计划使用我们的特殊显微镜来观察包含特殊序列的短DNA片段，这些序列指示RNA聚合酶分子从哪里开始读取特定的基因。DNA被标记有发出绿色或红色荧光的荧光染料；这些染料被放置在特定的点上，以便充当DNA结构变化的“记者”。在DNA中加入RNA聚合酶分子后，DNA的结构和环境会发生变化，这种变化可以通过荧光探针来感知；我们可以通过使用我们的特殊显微镜观察单个DNA分子来了解这些变化。事实上，我们可以直接观察到这些变化的发生，通过将DNA片段锚定在玻璃表面上，并通过记录转录的快速分子电影。我们还将开发数学模型，试图基于我们从分子电影中记录的数据来描述转录过程。这将使我们能够更好地了解这一过程的各个步骤，并对与其他基因和其他行为有关的其他DNA序列做出预测。这一努力将提高我们对基因表达如何工作的理解(基因打开或关闭的方式、时间和频率)，以及当基因表达障碍时为什么会出现某些疾病。我们在基因表达方面的工作也将有助于开发能够改善健康的新药。

The proposed work involves the development of special methods for ultra-sensitive microscopy and theoretical understanding of important genetic processes involved in gene expression. Gene expression is the path that leads from the genetic information (stored in DNA) to the manufacturing of proteins (the molecules that make up most of the machines and structures of living cells). Specifically, the work focuses on the process of gene transcription, which is performed by protein machines called RNA polymerases. These tiny biological machines read DNA and copy the information into a messenger molecule (messenger RNA). A special feature of the proposed work is that it is performed using a special microscope (a 'single-molecule fluorescence microscope'). This microscope is carefully designed to allow detection and monitoring of individual ('single') fluorescent molecules present in a detection zone (as opposed to conventional microscopes that require thousands or millions of molecules to be present in a detection zone). The single-molecule fluorescence microscope allows one to determine the number of parts that make up a biological machine, to measure how strong the parts bind to each other, how far apart the parts are spaced and at what orientation, and what are the movements of the parts when the tiny biological machine works. We plan to use our special microscope to observe short DNA pieces containing special sequences that instruct an RNA polymerase molecule where to start reading a specific gene. The DNA is labelled with fluorescent dyes that emit fluorescence of green or red colour; the dyes are placed at specific points in order to act as 'reporters' of any changes in the structure of DNA. Upon adding RNA polymerase molecules to the DNA, the structure and the environment of the DNA change and the change is sensed by the fluorescent probes; we can learn about these changes by observing the single DNA molecules using our special microscope. In fact, we can observe these changes directly as they occur by anchoring the DNA pieces on a glass surface and by recording fast 'molecular movies' of transcription. We will also develop mathematical models that try to describe the transcription process based on the data we record from our molecular movies. This will allow us to understand better the various steps of the process and to make predictions for other DNA sequences which are connected to other genes and other behaviours. This effort will improve our understanding of how gene expression works (how, when and how frequently genes are turned on or off), and why certain diseases arise when gene expression malfunctions. Our work on gene expression will also aid in the development of new drugs that will improve health.