Interplay of bacterial transcription and chromosome organisation in vivo

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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.