Show simple item record

dc.contributor.advisorLiu, Wensheen_US
dc.creatorOdoi, Keturahen_US
dc.date.accessioned2012-07-16T15:58:29Zen_US
dc.date.accessioned2012-07-16T20:31:10Z
dc.date.available2014-09-16T07:28:19Z
dc.date.created2012-05en_US
dc.date.issued2012-07-16en_US
dc.date.submittedMay 2012en_US
dc.identifier.urihttp://hdl.handle.net/1969.1/ETD-TAMU-2012-05-11037en_US
dc.description.abstractSystematic 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.en_US
dc.format.mimetypeapplication/pdfen_US
dc.language.isoen_USen_US
dc.subjectPyl, pyrrolysineen_US
dc.subjectPylRS, pyrrolysyl-tRNA synthetaseen_US
dc.subjectNAA, noncanonical amino aciden_US
dc.subjectBocK, Ntert-butyloxycarbonyl)-L-lysineen_US
dc.subjectaaRS, aminoacyl-tRNA synthetaseen_US
dc.subjectMjTyrRS, Methanococcus jannaschii tyrosyl-tRNA synthetaseen_US
dc.subjectT4 PNK, T4 polynucleotide kinaseen_US
dc.subjectAzF, para-azido-L-phenylalanineen_US
dc.subjectESI-MS, electrospray ionization mass spectrometryen_US
dc.subjectPCR, polymerase chain reactionen_US
dc.subjectsfGFP, superfolder green fluorescent proteinen_US
dc.subjectsfGFP134TAG, the sfGFP gene with an amber mutation at N134 and a 6×His tag at its C-terminusen_US
dc.subjectsfGFP134TGA,the sfGFP gene with an opal mutation at N134 and a 6×His tag at its C-terminusen_US
dc.subjectsfGFP134TAA,the sfGFP gene with an ochre mutation at N134 and a 6×His tag at its C-terminusen_US
dc.subjectsfGFP134AGGA,the sfGFP gene with an quadruple AGGA mutation at N134 and a 6×His tag at its C-terminusen_US
dc.subjectsfGFP2TAG134TAA, the sfGFP gene with an amber mutation at S2, an ochre mutation at N134, and a 6×His tag at its C-terminusen_US
dc.subjectsfGFP2TGA, the sfGFP gene with an opal mutation at S2 and a 6×His tag at its C-terminusen_US
dc.subjectIPTG, isopropyl -D-thiogalactopyranosideen_US
dc.subjectRF, release factoren_US
dc.subjectTrpRS, tryptophanyl-tRNA synthetaseen_US
dc.subjectArgRS, arginyl-tRNA synthetaseen_US
dc.subjectTrp, tryptophanen_US
dc.subjectLys, lysineen_US
dc.subjectGlu, glutamateen_US
dc.subjectGln, glutamineen_US
dc.subjectPhe, phenylalanineen_US
dc.subjectArg, arginine.en_US
dc.titleOrthogonality and Codon Preference of the Pyrrolysyl-tRNA Synthetase-tRNAPyl pair in Escherichia coli for the Genetic Code Expansionen_US
dc.typeThesisen
thesis.degree.departmentChemistryen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.grantorTexas A&M Universityen_US
thesis.degree.nameMaster of Scienceen_US
thesis.degree.levelMastersen_US
dc.contributor.committeeMemberBegley, Tadhgen_US
dc.contributor.committeeMemberRaushel, Franken_US
dc.contributor.committeeMemberWatanabe, Coranen_US
dc.type.genrethesisen_US
dc.type.materialtexten_US
local.embargo.terms2014-07-16en_US


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record