How Transcription Factor Affinity Influences Target Gene Transcription

Date
2017
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Volume Title
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Abstract
Transcriptional 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.
Description
Keywords
Genetics, Microbiology, Biology--Molecular, Biochemistry
Citation
Lancaster, 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/26397