Single Molecule Investigations of the Twin Supercoiling Domain Hypothesis
Transcription is the molecular process in the cell whereby the genetic information from DNA is copied into messenger RNA by the molecular motor RNA polymerase (RNAP) which catalyses the polymerisation of ribonucleotides. Since DNA is a helical molecule, the RNAP needs to rotate relative to the DNA template to undergo transcription. On a torsionally constrained template, the RNAP will therefore cause over-winding of the DNA in front of it and under-wound DNA behind, as it translocates along the DNA. This effect is known as the twin supercoiling domain hypothesis and it is expected that build-up of localised supercoiling within the DNA will affect the ability of the RNAP to copy the gene in question: it is therefore a fundamental physical mechanism that modulates gene expression.
We investigate this question through constructed systems in vitro using high resolution imaging of individual molecular complexes using atomic force microscopy (AFM). The translocation of DNA through RNAP as transcription occurs in vitro was first followed using atomic force microscopy (AFM) at the single molecule level (1,2). More recently, we have been investigating the interactions of more than one RNAP on a single DNA template (3, 4). Interestingly, we find that the position of one RNAP during transcription is influenced by another RNAP operating at the same time. Currently, it seems that this effect occurs regardless of whether these RNAPs are travelling towards each other or in the same direction. This project will continue our investigations into the fundamental mechanisms involved in spatial regulation of the RNAPs. Our working hypothesis is that local supercoiling of the DNA between RNAPs causes them to stall or pause when they get too close to one another. This may be one fundamental way that the cell controls gene expression through the physical properties of the DNA, but more work is needed to prove the hypothesis and understand the details.
It is being increasingly discovered that many genes lie in a nested formation, such that the promoters are convergently aligned on opposite DNA strands in the double helix. The implications for simultaneous expression of these genes are obvious and lead one to ask what would occur if two RNAP encounter each other on a single template (5). Collaborators in the School of Dentistry investigate a nested gene system involved in tooth enamel development where the expression of the biomineralising amelogenin protein may be compromised (6). The outcomes of this project will help to inform us about fundamental aspects of developmental biology and have long term impact on the treatment of diseased states associated with altered gene expression.
This is an interdisciplinary project suitable for a candidate with training in physics, chemistry or biochemistry or a related topic (or more specifically biophysics or physical biochemistry). See www.astbury.leeds.ac.uk for details of research in the Thomson group and related projects.
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