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Bacterial chromosome structure and transcription

细菌染色体结构和转录

基本信息

批准号：
BB/J006076/1
负责人：
Steve Busby
金额：
$ 66.83万
依托单位：
University of Birmingham
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2012
资助国家：
英国
起止时间：
2012 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FJ006076%2F1
关键词：
Bacterial chromosome structure transcription

项目摘要

Bacteria are microscopic free living organisms that are found nearly everywhere on earth, including in the human body. Their actions have big impacts on the environment at all levels and they also affect human health and happiness. Bacterial cells are organised in a different way to animal cells, notably with respect to how they handle their DNA. In animal cells, the DNA is packaged into individual chromosomes that are kept in a separate membrane-bound compartment of the cell called the nucleus. For most bacteria, their DNA consists of millions of base pairs in a single chromosome that is free in the main cell compartment. This creates a logistic problem since bacterial cells are small and, in order to fit the DNA into the cell, it has to be highly compacted by folding. Microscopy studies have shown that, in many bacteria, the chromosome is restricted to a part of the cell called the nucleoid. We are interested in how proteins interact with bacterial chromosome DNA in order to compact it into the nucleoid, and over a dozen different proteins that contribute to the compaction have now been identified. Whilst we understand the actions of many of these proteins when bound at individual DNA targets, we have little idea how these proteins act together on a bigger scale to organise DNA in the bacterial nucleoid.This proposal is prompted by the recent discovery of specific locations on the chromosome of a common bacterium, Escherichia coli, where the amount of bound protein is especially high. It has been suggested that these highly occupied targets act as the organising centres of the nucleoid by clustering together segments from different parts of the chromosome. It is thought that this clustering is essential to the compaction of the Escherichia coli chromosome and that similar mechanisms operate in most bacteria. Hence our aim is to identify the proteins that bind at these targets and start to build up a detailed protein occupancy map of the Escherichia coli chromosome. To achieve this, we will exploit a newly developed method called DNA sampling. Having identified the proteins that bind at different targets, we next want to build up a DNA proximity map by identifying chromosome segments that are far apart in the DNA sequence but clustered together in the 3-dimensional space of the nucleoid. One of the problems with doing this is that bacterial nucleoids are not fixed structures and each locus on the DNA may well make short-lived interactions with many other loci. Hence, to capture transient interactions, we will use a method called chromatin conformation capture, and, by combining it with high throughput sequencing, we will be able to record the different interactions. Taken together, this information will allow us to build up a picture of the different interactions that hold the Escherichia coli nucleoid together. Finally, we will investigate the possibility that the folding of gene DNA into a bacterial nucleoid affects its ability to be expressed. This is most likely because the folding restricts the accessibility of certain DNA elements that must be recognised by the proteins that initiate gene expression. We already have some preliminary data to show that this is the case for some of the regions of high protein binding. Hence, we are planning to use state-of-the-art fluorescence microscopy to find out where these transcriptionally silent loci are positioned in the nucleoid. These experiments will provide important information for modellers who want to predict patterns of expression from the DNA base sequence of any bacterium.

细菌是一种微观的自由生物体，几乎在地球上的任何地方都可以找到，包括人体。他们的行为对各个层面的环境都有很大的影响，也影响到人类的健康和幸福。细菌细胞的组织方式与动物细胞不同，特别是它们如何处理它们的DNA。在动物细胞中，DNA被包装成单独的染色体，这些染色体被保存在细胞的一个单独的膜结合区室中，称为细胞核。对于大多数细菌来说，它们的DNA由单个染色体中的数百万个碱基对组成，这些染色体在主细胞区室中是自由的。这就产生了一个逻辑问题，因为细菌细胞很小，为了将DNA装入细胞，它必须通过折叠高度压缩。显微镜研究表明，在许多细菌中，染色体被限制在细胞的一部分，称为类核。我们感兴趣的是蛋白质如何与细菌染色体DNA相互作用，以便将其压缩成类核，现在已经确定了十几种有助于压缩的不同蛋白质。虽然我们了解这些蛋白质结合在单个DNA靶点上时的作用，但我们对这些蛋白质如何在更大范围内共同作用以组织细菌类核中的DNA知之甚少，这一提议是由最近发现的一种常见细菌大肠杆菌染色体上的特定位置引起的，在该位置结合的蛋白质量特别高。有人提出，这些高度占据的靶点通过将来自染色体不同部分的片段聚集在一起而充当类核的组织中心。据认为，这种聚类是必不可少的压缩大肠杆菌染色体和类似的机制在大多数细菌中运作。因此，我们的目标是确定结合在这些目标的蛋白质，并开始建立一个详细的蛋白质占据图的大肠杆菌染色体。为了实现这一点，我们将利用一种新开发的称为DNA采样的方法。在确定了与不同靶点结合的蛋白质之后，我们接下来想通过识别在DNA序列中相距很远但在类核的三维空间中聚集在一起的染色体片段来构建DNA邻近图。这样做的一个问题是细菌类核不是固定的结构，DNA上的每个位点都可能与许多其他位点发生短暂的相互作用。因此，为了捕获瞬时相互作用，我们将使用一种称为染色质构象捕获的方法，并且通过将其与高通量测序相结合，我们将能够记录不同的相互作用。综合起来，这些信息将使我们能够建立一个不同的相互作用，使大肠杆菌类核在一起的图片。最后，我们将研究基因DNA折叠成细菌类核影响其表达能力的可能性。这很可能是因为折叠限制了某些DNA元件的可及性，而这些DNA元件必须被启动基因表达的蛋白质识别。我们已经有一些初步的数据表明，这是一些高蛋白结合区域的情况。因此，我们计划使用最先进的荧光显微镜来找出这些转录沉默位点位于类核中的位置。这些实验将为想要从任何细菌的DNA碱基序列预测表达模式的建模者提供重要信息。