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

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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.