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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.

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1

Orthogonality and Codon Preference of the Pyrrolysyl-tRNA Synthetase-tRNAPyl pair in Escherichia coli for the Genetic Code Expansion

Odoi, Keturah 2012 May 1900 (has links)
Systematic studies of basal nonsense suppression, orthogonality of tRNAPyl variants, and cross recognition between codons and tRNA anticodons are reported. E. coli displays detectable basal amber and opal suppression but shows a negligible ochre suppression. Although detectable, basal amber suppression is fully inhibited when a pyrrolysyl-tRNA synthetase (PylRS)-tRNAPyl_CUA pair is genetically encoded. trnaPyl_CUA is aminoacylated by an E. coli aminoacyl-tRNA synthetase at a low level, however, this misaminoacylation is fully inhibited when both PylRS and its substrate are present. Besides that it is fully orthogonal in E. coli and can be coupled with PylRS to genetically incorporate a NAA at an ochre codon, tRNAPyl_UUA is not able to recognize an UAG codon to induce amber suppression. This observation is in direct conflict with the wobble base pair hypothesis and enables using an evolved M. jannaschii tyrosyl-tRNA synthetase-tRNAPyl_UUA pair and the wild type or evolved PylRS-tRNAPyl_UUA pair to genetically incorporate two different NAAs at amber and ochre codons. tRNAPyl_UCA is charged by E. coli tryptophanyl-tRNA synthetase, thus not orthogonal in E. coli. Mutagenic studies of trnaPyl_UCA led to the discovery of its G73U form which shows a higher orthogonality. Mutating trnaPyl_CUA to trnaPyl_UCCU not only leads to the loss of the relative orthogonality of tRNAPyl in E. coli but also abolishes its aminoacylation by PylRS.
Pyl, pyrrolysine
PylRS, pyrrolysyl-tRNA synthetase
NAA, noncanonical amino acid
aaRS, aminoacyl-tRNA synthetase
T4 PNK, T4 polynucleotide kinase
AzF, para-azido-L-phenylalanine
PCR, polymerase chain reaction
RF, release factor
TrpRS, tryptophanyl-tRNA synthetase
ArgRS, arginyl-tRNA synthetase
Trp, tryptophan
Lys, lysine
Glu, glutamate
Gln, glutamine
Phe, phenylalanine
Arg, arginine.

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