Interplay of bacterial transcription and chromosome organisation in vivo

体内细菌转录和染色体组织的相互作用

• 批准号: BB/N018656/1
• 负责人: Achillefs Kapanidis
• 金额: $ 50.07万
• 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: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FN018656%2F1
• 关键词: Interplay; bacterial; transcription; chromosome; organisation

Our study uses ultra-sensitive microscopes to observe important processes in the gene expression, which is the path that leads from the genetic information (stored in DNA, the molecule that forms the chromosomes of living organisms) 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), and ensure that the right genes are expressed at the right time, the right place, and at the required level.Much of what we know about how RNA polymerase works to transcribe DNA to RNA comes from studies with purified proteins and DNA in the test tube; these involve simple mixtures of RNA polymerase with DNA sequences and accessory transcription proteins that can make transcription faster or slower. However, the mechanisms of transcription in actual living organisms and cells can be very different, due to the myriad of other biological components that are present in cells, and due to the way that the genes are packaged in the "bacterial nucleoid", which is a tightly packed structure made of the bacterial DNA and some of its proteins. An example of the complexity that characterises gene transcription in living cells is the fact that genes that are being transcribed appear to be on the surface of the nucleoid, and not buried deeply into it. Another example of complexity is that RNA polymerases seem to operate in large teams ("clusters"), with the number of team members and the location of the team depending on how many nutrients the cells have in their environment, and how fast they are growing.To study the process of gene transcription in its natural environment of living cells, and understand how this process is organised and controlled, we will use advanced fluorescence microscopy to look the position of labelled RNA polymerases and specific genes in living bacterial cells. We will use the bacterium Escherichia coli, which is a simple model organism for understanding biological mechanisms. A special feature of our 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 inside living cells (as opposed to conventional microscopes that require thousands or millions of fluorescent molecules).Using our powerful microscope to record movies of the position of individual genes and RNA polymerase molecules, we will see how genes change their position in the cell as they are being transcribed, and analyse the influence of other proteins that are known to control the amount of transcription in cells. We will also study how the individual RNA polymerase teams are organized, how many members the teams have, and how the teams come together or disband. Finally, we will study whether RNAP teams co-operate to work more efficiently, and if this is the case, we will examine how the teams assemble to achieve this high transcription efficiency. Our studies will improve our understanding of how gene expression works in living cells, and help other scientists to build more efficient artificial cells, as well as to develop new pharmaceuticals that will improve health by disabling the RNAP teams of dangerous microbes.

我们的研究使用超灵敏的显微镜来观察基因表达中的重要过程，这是从遗传信息（存储在DNA中，DNA是形成生物体染色体的分子）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中我们对RNA聚合酶如何将DNA转录为RNA的了解，大部分来自于对试管中纯化蛋白质和DNA的研究;这些涉及RNA聚合酶与DNA序列和辅助转录蛋白的简单混合物，其可以使转录更快或更慢。然而，由于细胞中存在无数其他生物组分，并且由于基因包装在“细菌类核”中的方式，实际活生物体和细胞中的转录机制可能非常不同，“细菌类核”是由细菌DNA及其一些蛋白质组成的紧密包装结构。活细胞中基因转录的复杂性的一个例子是，被转录的基因似乎位于类核的表面，而不是深埋其中。另一个复杂性的例子是RNA聚合酶似乎是以大团队的形式运作的（“集群”），团队成员的数量和团队的位置取决于细胞在其环境中有多少营养物质，为了研究在活细胞的自然环境中基因转录的过程，并了解这一过程是如何组织和控制的，我们将使用先进的荧光显微镜来观察标记的RNA聚合酶和特定基因在活细菌细胞中的位置。我们将使用细菌大肠杆菌，这是一个简单的模式生物了解生物机制。我们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。这种显微镜经过精心设计，可以检测和监测活细胞内的单个荧光分子（与传统的显微镜相反，传统的显微镜需要数千或数百万个荧光分子）。使用我们强大的显微镜记录单个基因和RNA聚合酶分子位置的电影，我们将看到基因在转录时如何改变它们在细胞中的位置，并分析其他已知控制细胞转录量的蛋白质的影响。我们还将研究单个RNA聚合酶团队是如何组织的，团队有多少成员，以及团队如何聚集或解散。最后，我们将研究RNAP团队是否合作以更有效地工作，如果是这样，我们将研究团队如何组装以实现这种高转录效率。我们的研究将提高我们对基因表达如何在活细胞中工作的理解，并帮助其他科学家建立更有效的人工细胞，以及开发新的药物，通过禁用RNAP团队的危险微生物来改善健康。

项目成果

RNA polymerase redistribution supports growth in E. coli strains with a minimal number of rRNA operons.

• DOI: 10.1093/nar/gkad511
• 发表时间: 2023-08-25
• 期刊: Nucleic acids research
• 影响因子: 14.9

Confinement-Free Wide-Field Ratiometric Tracking of Single Fluorescent Molecules

• DOI: 10.1016/j.bpj.2019.10.033
• 发表时间: 2019-12-03
• 期刊: BIOPHYSICAL JOURNAL
• 影响因子: 3.4
• 作者: Gilboa，Barak;Jing，Bo;Kapanidis，Achillefs N.
• 通讯作者: Kapanidis，Achillefs N.

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

• DOI: 10.1093/nar/gky482
• 发表时间: 2018-08-21
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Duchi D;Mazumder A;Malinen AM;Ebright RH;Kapanidis AN
• 通讯作者: Kapanidis AN

Single-molecule tracking reveals the functional allocation, in vivo interactions and spatial organization of universal transcription factor NusG

• DOI: 10.1101/2022.11.21.517430
• 发表时间: 2022-11
• 期刊: bioRxiv
• 作者: Hafez El Sayyed;Oliver J. Pambos;Mathew Stracy;M. Gottesman;A. Kapanidis

Understanding Protein Mobility in Bacteria by Tracking Single Molecules.

• DOI: 10.1016/j.jmb.2018.05.002
• 发表时间: 2018-10-26
• 期刊: Journal of molecular biology
• 影响因子: 5.6
• 作者: Kapanidis AN;Uphoff S;Stracy M
• 通讯作者: Stracy M