title: Locked and proteolysis-based transcription activator-like effector (TALE) regulation.
abstract: Development of orthogonal, designable and adjustable transcriptional regulators is an important goal of synthetic biology. Their activity has been typically modulated through stimulus-induced oligomerization or interaction between the DNA-binding and activation/repression domain. We exploited a feature of the designable Transcription activator-like effector (TALE) DNA-binding domain that it winds around the DNA which allows to topologically prevent it from binding by intramolecular cyclization. This new approach was investigated through noncovalent ligand-induced cyclization or through a covalent split intein cyclization strategy, where the topological inhibition of DNA binding by cyclization and its restoration by a proteolytic release of the topologic constraint was expected. We show that locked TALEs indeed have diminished DNA binding and regain full transcriptional activity by stimulation with the rapamycin ligand or site-specific proteolysis of the peptide linker, with much higher level of activation than rapamycin-induced heterodimerization. Additionally, we demonstrated reversibility, activation of genomic targets and implemented logic gates based on combinations of protein cyclization, proteolytic cleavage and ligand-induced dimerization, where the strongest fold induction was achieved by the proteolytic cleavage of a repression domain from a linear TALE.
authors.affiliation: Laboratory of Biotechnology, National Institute of Chemistry, Ljubljana 1000, Slovenia EN-FIST Centre of Excellence, Ljubljana 1000, Slovenia Graduate School of Biomedicine, University of Ljubljana, Ljubljana 1000, Slovenia.
authors.forename: Jan
authors.initials: J
authors.lastname: Lonzarić
authors.affiliation: Laboratory of Biotechnology, National Institute of Chemistry, Ljubljana 1000, Slovenia EN-FIST Centre of Excellence, Ljubljana 1000, Slovenia Graduate School of Biomedicine, University of Ljubljana, Ljubljana 1000, Slovenia.
authors.forename: Tina
authors.initials: T
authors.lastname: Lebar
authors.affiliation: Laboratory of Biotechnology, National Institute of Chemistry, Ljubljana 1000, Slovenia.
authors.forename: Andreja
authors.initials: A
authors.lastname: Majerle
authors.affiliation: Laboratory of Biotechnology, National Institute of Chemistry, Ljubljana 1000, Slovenia EN-FIST Centre of Excellence, Ljubljana 1000, Slovenia.
authors.forename: Mateja
authors.initials: M
authors.lastname: Manček-Keber
authors.affiliation: Laboratory of Biotechnology, National Institute of Chemistry, Ljubljana 1000, Slovenia EN-FIST Centre of Excellence, Ljubljana 1000, Slovenia roman.jerala@ki.si.
authors.forename: Roman
authors.initials: R
authors.lastname: Jerala
mesh_terms: D000595:Amino Acid Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: D004268:DNA-Binding Proteins; D015534:Trans-Activators; D004247:DNA