Several cellular processes in nature are directly dictated by gene expression. Transcription in eukaryotic cells is regulated through cooperative and synergistic actions of an ensemble of proteins, and deciphering the cooperative mechanisms could be used as a new method to design the regulation of cellular processes. Understanding this process would enable the design of intricate regulatory circuits in synthetic biology. This dissertation focuses on the effect transcription activator like effector (TALE) has on the activity of another transcription factor bound tens of base pairs away. The TALE protein, which lacks an activation domain, can enhance the effect of another transcription factor regardless of its orientation, its position upstream or downstream of the transcription factor binding site, and the distance between the respective binding sites. The effect of the TALE protein was demonstrated in several mammalian cell lines in combination with three structurally diverse DNA binding domains, regardless of whether they activate or repress transcription. The minimum and maximum distance between binding sites where the effect of TALE protein could be observed were determined. The first part of the dissertation describes a platform for investigating a potential synergistic effect of the TALE protein with diverse transcription factors, and providing mechanistic insights into cooperative interactions and the role of DNA in translation regulation. The discovered ability of TALE proteins to enhance transcriptional regulation of other transcription factors further expands the variety of the effects of TALE proteins on transcription, which could act as an additional modulator in regulating transcriptional pathways. Allosteric proteins have an interesting potential as tools for synthetic biology. In addition to expanding our understanding of the variety of synergistic effects on transcriptional regulation, a designable DNA binding protein with characterized cooperative properties could serve as a tool to investigate the mechanisms of synergistic transcriptional regulation and harvest its potential.
|
