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

体外和计算机中初始转录的单分子分析

• 批准号: BB/H01795X/1
• 负责人: Achillefs Kapanidis
• 金额: $ 51万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2010
• 资助国家: 英国
• 起止时间: 2010 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FH01795X%2F1
• 关键词: Single; molecule; analysis; initial; transcription

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.

拟议的工作涉及超灵敏显微镜的特殊方法的发展和参与基因表达的重要遗传过程的理论理解。基因表达是从遗传信息（存储在DNA中）到蛋白质（构成活细胞大部分机器和结构的分子）制造的路径。具体来说，这项工作的重点是基因转录的过程，这是由蛋白质机器称为RNA聚合酶。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。这项工作的一个特点是使用特殊的显微镜（“单分子荧光显微镜”）进行。该显微镜经过精心设计，允许检测和监测检测区中存在的单个（“单个”）荧光分子（与传统显微镜相反，传统显微镜需要数千或数百万个分子存在于检测区中）。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，测量部件彼此结合的强度，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。我们计划使用特殊的显微镜来观察含有特殊序列的短DNA片段，这些序列指示RNA聚合酶分子从何处开始阅读特定基因。DNA被荧光染料标记，发出绿色或红色的荧光;染料被放置在特定的点，以作为DNA结构中任何变化的“报告者”。在DNA中加入RNA聚合酶分子后，DNA的结构和环境发生变化，荧光探针可以检测到这种变化;我们可以通过使用特殊的显微镜观察单个DNA分子来了解这些变化。事实上，我们可以通过将DNA片段锚定在玻璃表面并记录快速转录的“分子电影”来直接观察这些变化。我们还将开发数学模型，试图根据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理解该过程的各个步骤，并对与其他基因和其他行为相关的其他DNA序列进行预测。这项工作将提高我们对基因表达如何工作的理解（基因如何、何时以及如何频繁地打开或关闭），以及为什么当基因表达出现故障时会出现某些疾病。我们在基因表达方面的工作也将有助于开发改善健康的新药。

项目成果

Internalization and observation of fluorescent biomolecules in living microorganisms via electroporation.

• DOI: 10.3791/52208
• 发表时间: 2015-02-08
• 期刊: Journal of visualized experiments : JoVE
• 作者: Aigrain L;Sustarsic M;Crawford R;Plochowietz A;Kapanidis AN
• 通讯作者: Kapanidis AN

Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes.

单细菌转录起始复合物中的构象异质性和气泡动力学。

• DOI: 10.1093/nar/gkx1146
• 发表时间: 2018-01-25
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Duchi D;Gryte K;Robb NC;Morichaud Z;Sheppard C;Brodolin K;Wigneshweraraj S;Kapanidis AN
• 通讯作者: Kapanidis AN

Non-covalent Single Transcription Factor Encapsulation Inside a DNA Cage

DNA 笼内非共价单转录因子封装

• DOI: 10.1002/ange.201207914
• 发表时间: 2013
• 期刊: Angewandte Chemie
• 作者: Crawford R

The RNA polymerase clamp interconverts dynamically among three states and is stabilized in a partly closed state by ppGpp

RNA 聚合酶夹在三种状态之间动态地相互转换，并通过 ppGpp 稳定在部分闭合状态

• DOI: 10.1101/278838
• 发表时间: 2018
• 作者: Duchi D

The switching mechanism of the bacterial rotary motor combines tight regulation with inherent flexibility.

• DOI: 10.15252/embj.2020104683
• 发表时间: 2021-03-15
• 期刊: The EMBO journal
• 作者: Afanzar O;Di Paolo D;Eisenstein M;Levi K;Plochowietz A;Kapanidis AN;Berry RM;Eisenbach M
• 通讯作者: Eisenbach M