Single-molecule analysis of initial transcription in vitro and in silico

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The proposed work involves the development of special methods for ultra-sensitive microscopy and theoretical understanding of important genetic processes involved in gene expression. Gene expression is the path that leads from the genetic information (stored in DNA) 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). A special feature of the proposed 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 present in a detection zone (as opposed to conventional microscopes that require thousands or millions of molecules to be present in a detection zone). The single-molecule fluorescence microscope allows one to determine the number of parts that make up a biological machine, to measure how strong the parts bind to each other, how far apart the parts are spaced and at what orientation, and what are the movements of the parts when the tiny biological machine works. We plan to use our special microscope to observe short DNA pieces containing special sequences that instruct an RNA polymerase molecule where to start reading a specific gene. The DNA is labelled with fluorescent dyes that emit fluorescence of green or red colour; the dyes are placed at specific points in order to act as 'reporters' of any changes in the structure of DNA. Upon adding RNA polymerase molecules to the DNA, the structure and the environment of the DNA change and the change is sensed by the fluorescent probes; we can learn about these changes by observing the single DNA molecules using our special microscope. In fact, we can observe these changes directly as they occur by anchoring the DNA pieces on a glass surface and by recording fast 'molecular movies' of transcription. We will also develop mathematical models that try to describe the transcription process based on the data we record from our molecular movies. This will allow us to understand better the various steps of the process and to make predictions for other DNA sequences which are connected to other genes and other behaviours. This effort will improve our understanding of how gene expression works (how, when and how frequently genes are turned on or off), and why certain diseases arise when gene expression malfunctions. Our work on gene expression will also aid in the development of new drugs that will improve health.