Intracellular detection and functional impact of protein synthesis errors in S. cerevisiae and E. coli

Accurate translation of the mRNA code is vital for both proper cell function, and the production of functional recombinant protein products. Errors in translation, i.e. the incorporation of amino acids other than those that should be incorporated according to the genetic code, negatively affect the function of proteins. This thesis focuses upon two main areas.

1. Possible error detection pathways in S. cerevisiae are examined through the use of knockout strains and inhibitor treatments upon both optimised and dis-optimised GST genes. None of the surveillance pathway mutants used in this study showed detectable changes in the expression levels or in the detectable degradation products expressed from the recombinant GST genes, indicating that these pathways are not involved in detecting translational error products. However, the sensitivity of the western blotting techniques used in this study is limited, and possible alternate approaches are discussed.

2. An MS-based analysis of translational errors and their effects upon binding affinity in GST was carried out prior to this project. The results are examined herein through computational analysis, based upon factors such as 3D modelling, evolutionary conservation, and predicted tRNA ratios. Additionally, several translational error products were re-created as DNA mutants in order to allow experimental analysis of the effects of errors on protein function. These two approaches demonstrate the importance of protein folding upon error observation.