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

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.

This proposal describes single-molecule studies of the initial steps of gene transcription, from the point that RNA polymerase (the main transcriptional machinery) first interacts with promoter DNA to the point that it leaves the promoter and the promoter-proximal region. This phase of transcription is often where most of gene regulation occurs both in prokaryotes and eukaryotes; therefore it is vital to understand its mechanisms and determinants. Building on a large body of genetic, biochemical and structural work, we seek to use the single-molecule approach to recover the real-time dynamics of transcription and directly observe behaviour heterogeneity of potential biological significance. We will achieve this by using fluorophores incorporated mainly on DNA in a way that, upon interaction with RNA polymerase, a single-molecule fluorescence observable is generated. The observable will mainly be a change in the efficiency of fluorescence resonance energy transfer (FRET), which will report on conformational changes within transcription complexes immobilized on a solid support and imaged using a high-end detection path. We will use this in vitro approach to examine the mechanism of the abortive initiation of transcription and to evaluate the importance of promoter-like DNA sequences in the initial transcribed region of a gene; these sequences may be related to promoter escape. We will also build theoretical kinetic and stochastic models to help evaluate our results, understand the mechanism of early transcription and to predict promoter behaviour; this approach should be generalisable to large families of promoters. We anticipate many biologically important observations from this work, as well as development of several experimental and theoretical tools that will benefit biophysicists and life scientists alike.

We aim to develop real-time in vitro single-molecule assays for detecting initial transcription. To achieve this, we have been developing an ultrasensitive fluorescence microscope that detects single molecules of proteins and DNA. The description of the microscope will be useful for scientists and engineers who develop ultra-sensitive biosensing platforms based on fluorescence detection of modified biomolecules that possess specificity and selectivity to recognize analytes of interest. We also develop efficient software algorithms to process large data sets, a task that requires advanced data management and storage skills. Researchers involved in these tasks will acquire important transferable skills desirable in environments involving demanding image analysis and data processing. Extending temporal and spatial resolution requires novel instrumentation and software. Apart from the training benefit of such efforts, such development may be useful in many settings: in undergraduate practical courses, in a clinical diagnostics lab, or in a pharmaceutical company. Part of our work is inextricably linked with understanding photophysics, which are spectroscopic transitions related to the ability of fluorophores to absorb and emit light. Such characterization is important in order to interpret our signals; the photophysics of many fluorophores are complex and new behaviours often emerge at the single-molecule level. For example, switching between bright and dark states of a fluorophore has been used for super-resolution imaging, which has created a revolution in optical microscopy by shuttering the diffraction limit. The contribution of super-resolution imaging instruments to biology and biomedical science is expected to be substantial. Since our work involves real-time detection of a polymerase-based reaction using fluorescence, it is highly relevant to third-generation DNA sequencing, which sequences single DNA molecules. The ability to collect more photons and assign fluorescence states more quickly will result in faster reads, whereas the ability to work with a processive protein such as RNA polymerase will provide longer reads. Since many RNA polymerases can operate on DNA, it may be possible to obtain multiple reads on the same DNA. Thus, the proposed work may contribute to a technology that can replicate the success of Solexa in 2nd generation sequencing, leading to new jobs for the construction of equipment, reagents, consumables and software for single-molecule sequencing, and promote inward investment. One process central to the proposed work is abortive initiation; crucially, blocking abortive initiation by the antibiotic rifampicin is the main strategy for treating tuberculosis, an infectious disease that causes 1.6 million deaths per year worldwide; tuberculosis is unfortunately expected to become more widespread due to urbanization and overcrowding in the developing world, and due to the emergence of antibiotic resistance. Since the rifampicin activity on initial transcription is detected by our assays, we may use them for finding compounds that block various stages of transcription, increasing the synergistic impact of the drugs. Our assays can also be implemented on other nucleic acid polymerases involved in disease: e.g., HIV reverse transcriptase (a major for anti-HIV treatments). We will also use theoretical modeling to describe initial transcription in bacterial promoters. Modeling gene expression in such systems may be relevant in bionanotechnology and synthetic biology efforts that develop artificial cells. Finally, the biochemist involved in the proposed work will interact with researchers from a physical-science background and work on a complex problem using tools from many disciplines. This training of highly interdisciplinary nature will be essential for contributing to biotech companies that work on similar problems but focus on more technological aspects with biomedical importance.