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Title: Importance of single molecular determinant in bacterial tryptophanyl-tRNA synthetase fidelity in expanded genetic code
Author: Simova, Zuzana
ISNI:       0000 0004 2752 0276
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2011
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Nonnatural amino acid incorporation is a valuable method for introducing novel chemical functional groups into proteins. For this method, an orthogonal arninoacyl-tRNA synthetase (AARS) and a cognate tRNA that suppress an encoded stop codon are introduced into the cell (these components are required to be orthogonal). Nonnatural amino acids (NAAs) are usually incorporated efficiently by using Methanocaldococcus jannaschii tyrosyl-AARS/tyrosyl-tRNA pair (Mj TyrRS/A// tRNATyr) in Escherichia coli. High translation fidelity of a synthetase is achieved by site-directed mutagenesis of the competent active site residues. The active site of the mutant Mj TyrRS displays two crucial mutations of residues that interact with the tyrosine hydroxyl group (-OH). We demonstrated that the fidelity of the synthetase would be affected if only one of these residues is restored and does not undergo mutagenesis. We found a similar situation in the case of tryptophanyl-AARS (TrpRS) from Bacillus subtilis. TrpRSs are structurally similar to TyrRSs, but there is one crucial residue of the substrate specificity. We uncovered that a NAA system developed to incorporate 5-hydroxytryptophan (5-OH Tip) in mammalian cells does not contain this crucial residue mutation in the TrpRS active site. Even though this mutant TrpRS was designated as a high fidelity enzyme, our results challenge this conclusion. Expanded genetic codes have a similar capacity to impact science as has standard mutagenesis. Only the full impact of the method will be achieved if the technology functions in all cell types. Therefore, our reinvestigation of the first report of expanded genetic code in mammalian system is critical to ensuring that the field is on the optimum path to realising the full potential of the method.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available