Single-molecule analysis of transcription-elongation regulation mechanisms in living bacteria

我们的研究使用超灵敏显微镜来观察基因表达过程中发生的重要过程，即将存储在DNA中的遗传信息（以染色体的形式存在于细胞中）转化为制造蛋白质和其他分子的途径，这些分子作为活细胞的机械、传感器和结构框架。具体来说，这项工作的重点是基因转录的过程，这是由称为RNA聚合酶的蛋白质机器完成的，它读取DNA并将信息复制到RNA分子中。RNA既可以作为信息（即信使RNA或“mRNA”），也可以成为其他大型机器的一部分，如核糖体RNA或“rRNA”，它是核糖体的一部分，核糖体是细胞中制造蛋白质的机器。转录进一步由称为转录调节因子的蛋白质控制，确保正确的基因在正确的时间、正确的地点和所需的水平上表达。特别是，我们正在研究转录调节因子NusG和RfaH，它们在转录延伸阶段将RNA聚合酶与其他机器（如核糖体）偶联，RNA聚合酶在此阶段迅速扩展RNA分子。NusG和RfaH是非常重要的调节因子，因为它们形成的家族控制着所有生物体的转录；RfaH在生物医学上也很重要，因为它允许许多致病菌开启可能导致疾病的基因，并帮助逃避抗生素治疗。我们对RNA聚合酶和转录调节因子如何工作的了解大部分来自于对试管中纯化蛋白质和DNA的研究；这些包括RNA聚合酶与DNA序列的简单混合物以及可以加速或减缓转录的转录调节因子。然而，实际生物体和细胞中的转录机制可能非常不同，这既是由于细胞中存在无数其他生物成分，也是由于基因被包装在“细菌类核”中的方式，细菌类核是由细菌DNA和一些蛋白质组成的紧密排列的结构。复杂性的另一个例子是RNA聚合酶和一些转录因子似乎在大型团队（“集群”）中起作用，团队成员的数量和团队的位置取决于细胞在其环境中的营养物质，以及它们生长的速度。为了研究活细胞自然环境中的转录延伸，并了解这一过程是如何组织和控制的，我们使用先进的荧光显微镜来观察活细菌细胞中荧光标记的转录调节因子的位置、流动性和结构。我们主要使用大肠杆菌，一种简单的模式生物来理解生物学机制。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（“单个”）荧光分子（与需要数千或数百万荧光分子的传统显微镜相反）。使用我们强大的显微镜来记录NusG和RfaH调节分子的位置和运动，我们将看到它们如何在细胞中移动，识别它们的目标，并与其他机制相互作用以控制RNA延伸。我们还将使用荧光方法作为分子标尺来观察这些调节因子如何改变其形状和结构以控制转录的编排。最后，我们将测试同事确定的特定化学物质是否可以阻止RfaH的功能，从而作为一类新的抗生素。我们的研究将提高我们对基因表达如何在活细胞中起作用的理解，并帮助其他科学家更好地理解这些复杂的过程，构建更好的人造细胞，并开发新的抗生素。

Our study uses ultra-sensitive microscopes to observe important processes occurring during gene expression, the path that converts genetic information stored in DNA (present in cells in the form of chromosomes) to the manufacturing of proteins and other molecules that serve as the machinery, sensors, and structural framework of living cells. Specifically, the work focuses on the process of gene transcription, which is performed by protein machines called RNA polymerases, which read DNA and copy the information into RNA molecules. RNA can serve either as a message (i.e., messenger RNA or "mRNA") or become part of other large machinery, such as ribosomal RNA or "rRNA", which is part of the ribosomes, the machines that make proteins in the cell. Transcription is further controlled by proteins known as transcriptional regulators, ensuring that the right genes are expressed at the right time, the right place, and at the required level.In particular, we are studying transcriptional regulators NusG and RfaH, which couple RNA polymerase to other machineries (such as the ribosome) during the phase of transcription elongation, where the RNA polymerase rapidly extends RNA molecules. NusG and RfaH are very important regulators since the family they form controls transcription in all living organisms; RfaH is also a biomedically important, since it allows many pathogenic bacteria to turn on genes that can cause disease and help evade antibiotic treatments. Much of what we know about how RNA polymerase and transcription regulators work comes from studies with purified proteins and DNA in the test tube; these involve simple mixtures of RNA polymerase with DNA sequences and transcription regulators that can accelerate or slow down transcription. However, the mechanisms of transcription in actual living organisms and cells can be very different, both due to the myriad of other biological components present in cells, and due to the way that genes are packaged in the "bacterial nucleoid", a tightly packed structure made of the bacterial DNA and some of its proteins. Another example of complexity is that RNA polymerases and some transcription factors seem to operate in large teams ("clusters"), with the number of team members and the location of the team depending on the nutrients the cells have in their environment, and how fast they are growing.To study transcription elongation in its natural environment of living cells, and understand how this process is organised and controlled, we use advanced fluorescence microscopy to look the position, mobility, and structure of fluorescently labelled transcription regulators in living bacterial cells. We mainly use the bacterium Escherichia coli, 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 positions and the motions of molecules of the NusG and RfaH regulators, we will see how they move in the cell, recognise their targets, and interact with other machinery to control RNA elongation. We will also use a fluorescence method that acts as a molecular ruler to look at the choreography of how these regulators change their shapes and structures to control transcription. Finally, we will test whether specific chemicals identified by colleagues can stop the function of RfaH and thus act as a new class of antibiotics. Our studies will improve our understanding of how gene expression works in living cells, and help other scientists to understand better these complex processes, to build better artificial cells, and to develop new antibiotics.