A Novel TetR-Regulating Peptide Turns off rtTA-Mediated Activation of Gene Expression

Abstract

Conditional regulation of gene expression is a powerful and indispensable method for analyzing gene function. The “Tet-On” system is a tool widely used for that purpose. Here, the transregulator rtTA mediates expression of a gene of interest after addition of the small molecule effector doxycycline. Although very effective in rapidly turning on gene expression, the system is hampered by the long half-life of doxycycline which makes shutting down gene expression rapidly very difficult to achieve. We isolated an rtTA-binding peptide by in vivo selection that acts as a doxycycline antagonist and leads to rapid and efficient shut down of rtTA-mediated reporter gene expression in a human cell line. This peptide represents the basis for novel effector molecules which complement the “Tet-system” by enabling the investigator to rapidly turn gene expression not just on at will, but now also off.

The induction mechanism of the flavonoid‐responsive regulator FrrA

“…This remarkably simple allosteric mechanism, which is likely to play a role in many other TetR-type repressors, is particularly well-suited for regulatory circuits requiring a broad inducer specificity, as it hardly places any constraints on the precise chemical nature of the ligand. Given that their specificity can be altered without affecting the allosteric mechanism, TetR-type repressors are highly attractive as molecular switches in biotechnological applications and synthetic biology [37,38].…”

Procaspase activating compound 1 controls tetracycline repressor-regulated gene expression system

“…The TetR-regulated system is a valuable tool to control gene expression both in vitro and in vivo [1–3]. Although numerous effectors have been developed for this system [12–16], only four effectors have been reported to reverse Tc-induced gene expression [12,13,16]. GR33076X, a Tc homolog is a small-molecule antagonist acting on tTA in bacterial cells, but its effect was shown only at 100- to 1000-fold higher concentration than Tc [12].…”

“…Although TAP1 and TCP1 as TetR-regulating peptides have antagonistic activities in the presence of non-saturating amount (0.03 μM) of Dox [13], usage of the peptides is inconvenient for the TetR-regulated system because transformation of plasmids harboring the peptides and addition of IPTG are needed to express them in cells. Schmidt et al reported K79, which is a peptide switching off gene expression mediated by rtTA, but its effect was apparent only after treatment with it for 10 h [16]. Thus, antagonists with a better efficacy and a faster mode-of-action need to be developed for the TetR-regulated system.…”

“…To accomplish rapid turn-on/off of target gene expression, developing effectors acting like a Tc antagonist is required. TAP1, TCP1, and K79 peptides were discovered as Tc antagonists using the yeast two-hybrid method [13,16]. Goeke et al have reported that TAP1 and TCP1 peptides were Tc antagonists interacting with TetR residues binding the C- or D-rings of Tc [13].…”

Utilization of a Strongly Inducible DDI2 Promoter to Control Gene Expression in Saccharomyces cerevisiae

“…The tetracycline regulatory systems (Tet-on and Tet-off), which were initially found from the bacterial transcription factor Tet repressor (TetR) combined with a TetR-responsive promoter ( Berens and Hillen, 2003 ; Schmidt et al, 2014 ), have been widely applied to regulate gene expression in eukaryotes ( Baron and Bujard, 2000 ). In the Tet-off system the target gene expression is suppressed in the presence of tetracycline (Tet) or its derivative doxycycline (Dox) and can be activated by tTA, a Tet-controlled transactivator protein, binding to a Tet-responsive promoter element (TRE) in the absence of Tet or Dox ( Freundlieb et al, 1997 ; Baron and Bujard, 2000 ).…”