Translational efficiency is dictated in part by the availability of charged transfer RNA. Depletion of aminoacylated tRNAs (e.g. during recombinant protein expression) can increase translational errors and associated stress responses. Here, the role of tRNAs as regulators of gene expression was explored through development of synthetic, tRNA-regulated gene circuits, and through an investigation of the impact of tRNA aminoacylation on endogenous gene expression. Synthetic gene circuits initially explored the use of dominant negative alleles of the release factor eRF1 to modulate stop codon readthrough and translationally regulate gene expression. Mutant eRF1 proteins exhibited only a six-fold stimulatory effect on stop codon readthrough. The dominant negative phenotype was rescued partially by overexpression of eRF1, but not eRF3. Ultimately the severity of growth inhibition by these eRF1 alleles limited their utility in synthetic gene circuit design. A novel synthetic circuit was then implemented that utilised TetR interaction with a TetR-inducing peptide in order to control the expression of a suppressor tRNA, and thus a luciferase reporter gene. Using a parameterised mathematical model, the promoter configuration of the circuit was successfully optimised, allowing suppressor tRNAs to regulate the production of luciferase in both feedforward and positive feedback modes of operation. The effects of charged tRNA levels on the global translation network were dissected by regulating the S.cerevisiae glutamine tRNA synthetase gene GLN4 using a tet-off doxycyclineregulated promoter. tRNA synthetase depletion caused the activation of the Gcn4 amino acid starvation response due to accumulation of uncharged glutamine tRNAs. Doxycycline GLN4 shut-off caused increased amino acid production, and decreased ribosome biosynthesis at the transcriptomic and proteomic level, and further physiological changes proposed to result from compromised translation of glutamine-rich regulatory proteins. tRNA overexpression in the GLN4 depletion strain successfully caused altered competition between different isoacceptor tRNA types for their cognate synthetase resource. Together, these results support a growing understanding of tRNA as a key modulator of translation and gene expression in synthetic and natural systems.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:715449 |
| Date | January 2016 |
| Creators | McFarland, Matthew R. |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=231855 |
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