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Title: Evolution and in vitro analysis of orthogonal ribosomes
Author: Barrett, O. M.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2009
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In Part A of this project, I insert an RNA affinity tag into the 16S rRNA of O-ribosomes and demonstrate this does not affect their activity. I optimise the purification of O-ribosomes from the mixture of ribosomes in cells, using the affinity tag. I demonstrate that the strategy can be used to purify evolved O-ribosomes and the utility of the purification method by developing an in vitro RF1 termination assay on O-ribosomes. Using this assay I characterise, in vitro, a O-ribosome previously evolved for enhanced unnatural amino acid incorporation in response to an amber codon (Ribo-X). I demonstrate that Ribo-X has a decreased RF1 mediated termination with respect to the progenitor O-ribosome – providing a mechanistic explanation for our in vivo observations. The aim of Part B of this project was to further engineer the orthogonal mRNA-30S complex to prevent cross-reaction with wild type 50S and then to develop a mutant 50S subunit to specifically interact and function with the orthogonal mRNA-30S complex. This would create a fully orthogonal ribosome. A fully orthogonal ribosome would be fully decoupled from translating the proteome. This would set the stage for evolving the large subunit of the ribosome, in particular the PTC to expand the chemical scope of translation. Approaches based on analysis of sequence variation across species and the use random libraries selected O-30S mutants with reduced translational activity with the endogenous 50S. It proved impossible to functionally complement these O-30S mutants with mutant 50S. However, these experiments produced some interesting mutant ribosomes which highlighted possible future approaches to producing a fully orthogonal ribosome.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available