Download Atomic force microscopy studies of the molecular mechanisms of

School of Dentistry PhD Project Proposal Form 2017 entry
Project Title
Atomic force microscopy studies of the molecular mechanisms of
nested genes related to tooth formation
Lead
Supervisor
Dr Neil Thomson
CoSupervisor(s)
Dr William Bonass; Dr Georg Feichtinger
Project Summary
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.
Aims & Objectives
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 and more recently, we have
been investigating the interactions of more than one RNAP on a single DNA template.
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. We propose to
investigate a nested gene system involved in tooth enamel development where the
expression of the biomineralising amelogenin protein may be compromised. 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.
References (optional)