Riboswitches are regulatory structures present in the 5′-UTR of a wide range of bacterial mRNAs. They consist of a small-molecule binding aptamer domain, which affects the conformation of a nearby expression platform to control gene expression through a transcriptional or translational mechanism. Because of their ability to bind selectively to very small concentrations of ligand, in a protein-independent manner, they have great potential for use as novel small-molecule controllable gene expression systems. This thesis describes how a combination of chemical genetics and genetic selection were used to develop and test a novel riboswitch-based gene-expression system. Several constructs were generated which could respond in vivo to a variety of non-natural small heterocyclic compounds and output via a simple fluorescence based assay in a dose-dependent manner. Methods for controlling the overall protein expression landscape of the riboswitch-based gene-expression system are outlined. In addition, the rational design of mutant riboswitch aptamers with improved ligand-binding capabilities is described alongside attempts to modulate the structural stability of the expression platform. Riboswitches need to be highly discriminatory to function effectively in vivo, binding to one ligand from a cellular pool of thousands. Mutant riboswitches were created that responded specifically to the ligands ammeline or azacytosine, and were found to have no induction in the presence of adenine, the wild-type riboswitch ligand. This in vivo ligand orthogonality was confirmed by subsequent in vitro studies. The ligand-induced structural changes undertaken by the mutant riboswitch aptamer domains were subsequently characterised using a variety of in vitro methods including SHAPE, ITC and x-ray crystallography. Finally, the feasibility of using riboswitch gene-expression systems in fully synthetic applications was demonstrated through the construction and analysis of small synthetic gene clusters and operons. The in vivo expression of two fluorescent proteins under independent riboswitch control was studied under single and dual induction for a range of ligand concentrations. The ability to control the expression of multiple genes is highly desirable in the emerging field of synthetic biology, the results described here indicate that riboswitches are ideally suited to complement current gene expression tools.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:686704 |
| Date | January 2011 |
| Creators | Duncan, John Nichlaus |
| Contributors | Micklefield, Jason |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/coevolution-of-small-molecule-responsive-riboswitches-by-chemical-and-genetic-selection(9f0be71a-519d-4766-b862-3b38cfcc57cd).html |
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