How Transcription Factor Affinity Influences Target Gene Transcription

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atmire.migration.oldid5979
dc.contributor.advisorMcGhee, James
dc.contributor.authorLancaster, Brett
dc.contributor.committeememberGrewal, Savraj
dc.contributor.committeememberBrook, William
dc.contributor.committeememberHansen, David
dc.contributor.committeememberStormo, Gary
dc.date.accessioned2017-09-20T15:25:10Z
dc.date.available2017-09-20T15:25:10Z
dc.date.issued2017
dc.date.submitted2017en
dc.description.abstractTranscriptional activation is a complex multifactorial process that centres on two fundamental properties of transcription factor proteins: sequence specific DNA binding and the subsequent recruitment of RNA polymerase to a target gene promoter. I have established an experimental system to investigate, inside the living animal, how transcription factor binding affinity at a promoter quantitatively influences transcriptional activity of a target gene. In C. elegans, the major intestinal transcription factor ELT-2 binds to a core TGATAA motif. The intestine-specific asp-1 protease gene is a direct target of ELT-2 and is controlled by two TGATAA sites. Two versions of asp-1 were created by introducing a novel KpnI restriction site at different locations in the coding region, thereby producing two reporters distinguishable by restriction digestion. The two reporters are combined into multicopy extrachromosomal transgenic arrays in the same animal, one reporter controlled by a wildtype promoter, the other by a promoter with altered ELT-2 binding sites. RNA isolation followed by RT-PCR, restriction digestion, and electrophoresis, allows the levels of both reporter transcripts to be measured simultaneously. We have measured relative affinities of ELT-2 for TGATAA variants in vitro by competitive band shifts, by both direct competition of two target sequences and by high-throughput competition of a library of target sequences (Spec-seq). As an example, the in vitro ELT-2 binding affinity to the core sequence CATGATAATC is ten-fold lower than to ACTGATAAGA. When this low affinity sequence is introduced into both of two TGATAA sites of the asp-1 promoter, the level of asp-1 transcripts measured in vivo are reduced five-fold. Overall, the relation between transcription factor binding affinity at the promoter and mRNA production by the target gene appears monotonic until plateauing at maximum output.en_US
dc.identifier.citationLancaster, B. (2017). How Transcription Factor Affinity Influences Target Gene Transcription (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/26397en_US
dc.identifier.doihttp://dx.doi.org/10.11575/PRISM/26397
dc.identifier.urihttp://hdl.handle.net/11023/4111
dc.language.isoeng
dc.publisher.facultyGraduate Studies
dc.publisher.institutionUniversity of Calgaryen
dc.publisher.placeCalgaryen
dc.rightsUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.
dc.subjectGenetics
dc.subjectMicrobiology
dc.subjectBiology--Molecular
dc.subjectBiochemistry
dc.subject.otherTranscription
dc.subject.otherGene Regulation
dc.titleHow Transcription Factor Affinity Influences Target Gene Transcription
dc.typedoctoral thesis
thesis.degree.disciplineBiochemistry and Molecular Biology
thesis.degree.grantorUniversity of Calgary
thesis.degree.nameDoctor of Philosophy (PhD)
ucalgary.item.requestcopytrue
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