Feedback Control of Protein Expression in Mammalian Cells by Tunable Synthetic Translational Inhibition

Abstract

Feedback regulation plays a crucial role in dynamic gene expression in nature, but synthetic translational feedback systems have yet to be demonstrated. Here we use an RNA/protein interaction-based synthetic translational switch to create a feedback system that tightly controls the expression of proteins of interest in mammalian cells. Feedback is mediated by modified ribosomal L7Ae proteins, which bind a set of RNA motifs with a range of affinities. We designed these motifs into L7Ae-encoding mRNA. Newly translated L7Ae binds its own mRNA, inhibiting further translation. This inhibition tightly feedback-regulates the concentration of L7Ae and any fusion partner of interest. A mathematical model predicts system behavior as a function of RNA/protein affinity. We further demonstrate that the L7Ae protein can simultaneously and tunably regulate the expression of multiple proteins of interest by binding RNA control motifs built into each mRNA, allowing control over the coordinated expression of protein networks.

Controllable molecular motors engineered from myosin and RNA

“…Guided by crystal structures of L7Ae-RNA 22 and of myosin VI 23,24 , we aligned the terminal helices and optimized the phasing of the junction to orient a bound kink-turn motif as a structural foundation from which to build extended RNA lever arms. The interaction of L7Ae with kink-turn motifs has been previously exploited in nanotechnology and synthetic biology applications 25–27 , and yields stable complexes with reported K d values of ~1 nM and dissociation rates of ~2–7 × 10 −4 s −1 25,27,28 .…”

Engineering Gene Circuits for Mammalian Cell–Based Applications

“…This simple approach was first shown using the L7Ae protein-C/D box RNA motif, which was engineered into reporter mRNA transcripts. In subsequent studies, further advancements have been made to tune input -output functions and to use multiple protein-responsive aptamers for the robust control of endogenous pathways and gene circuits (Saito et al 2011;Goldfless et al 2012;Stapleton et al 2012;Endo et al 2013). In a similar approach, protein-binding aptamers (MS2, p65, p50, and b-catenin aptamers) have been introduced into the proximity of splicing recognition sites of an alternative exon element of mRNA precursors (Fig.…”

Biomolecular implementation of a quasi sliding mode feedback controller based on DNA strand displacement reactions

“…Nominal Values S qsm total substrate 4 nM X 3 tuning parameter 0.1 nM k b 1 forward binding rate 10 7 /M/s k b 2 forward binding rate 10 7 /M/s k c 1 catalytic reaction rate 100 q i C max /s k c 2 catalytic reaction rate 50 q i C max /s Table III and the initial concentrations of the non-auxiliary species in equations (23)-(28) are set to zero, i.e. X 2 0 = A 0 = 0 nM.…”