Bacterial chromosome structure and transcription

细菌是一种微小的自由生命有机体，几乎在地球上的任何地方都可以找到，包括在人体内。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，特别是在处理DNA方面。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中一个单独的被膜束缚的隔间，称为细胞核。对于大多数细菌来说，他们的DNA由数百万个碱基对组成，存在于单个染色体中，该染色体在主细胞室中是自由的。这就产生了一个逻辑问题，因为细菌细胞很小，为了让DNA适合细胞，它必须通过折叠高度紧凑。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，以便将其压缩到类核中，现在已经确定了十几种不同的蛋白质，这些蛋白质有助于压缩。虽然我们了解其中许多蛋白质在与个别DNA靶标结合时的作用，但我们对这些蛋白质如何在更大范围内共同作用以在细菌核仁中组织DNA的作用知之甚少。这一建议是由最近在一种常见细菌--大肠杆菌的染色体上发现的特定位置推动的，在那里结合蛋白的数量特别高。有人认为，这些高度占据的目标通过将来自染色体不同部分的片段聚集在一起，充当类核的组织中心。据认为，这种聚集对大肠杆菌染色体的紧凑是必不可少的，类似的机制在大多数细菌中都有应用。因此，我们的目标是鉴定结合在这些靶点上的蛋白质，并开始建立一个详细的大肠杆菌染色体蛋白质占有图。为了实现这一点，我们将利用一种名为DNA采样的新方法。在确定了与不同靶点结合的蛋白质后，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建立DNA接近图。这样做的一个问题是，细菌的类核并不是固定的结构，DNA上的每个基因座很可能与许多其他基因座进行短暂的相互作用。因此，为了捕捉瞬时相互作用，我们将使用一种称为染色质构象捕捉的方法，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。综上所述，这些信息将使我们能够建立一幅将大肠杆菌类核糖体结合在一起的不同相互作用的图景。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元件的可获得性，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的基因座在类核中的位置。这些实验将为想要根据任何细菌的DNA碱基序列预测表达模式的模型师提供重要信息。

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.