Engineering ligand-responsive RNA controllers in yeast through the assembly of RNase III tuning modules

Abstract

The programming of cellular networks to achieve new biological functions depends on the development of genetic tools that link the presence of a molecular signal to gene-regulatory activity. Recently, a set of engineered RNA controllers was described that enabled predictable tuning of gene expression in the yeast Saccharomyces cerevisiae through directed cleavage of transcripts by an RNase III enzyme, Rnt1p. Here, we describe a strategy for building a new class of RNA sensing-actuation devices based on direct integration of RNA aptamers into a region of the Rnt1p hairpin that modulates Rnt1p cleavage rates. We demonstrate that ligand binding to the integrated aptamer domain is associated with a structural change sufficient to inhibit Rnt1p processing. Three tuning strategies based on the incorporation of different functional modules into the Rnt1p switch platform were demonstrated to optimize switch dynamics and ligand responsiveness. We further demonstrated that these tuning modules can be implemented combinatorially in a predictable manner to further improve the regulatory response properties of the switch. The modularity and tunability of the Rnt1p switch platform will allow for rapid optimization and tailoring of this gene control device, thus providing a useful tool for the design of complex genetic networks in yeast.

Tuning Response Curves for Synthetic Biology

“…By introducing rationally designed hairpins into E. coli mRNA, Carrier and Keasling 119 were able to influence half-lives over an order-of-magnitude range. More recently, Babiskin and Smolke 120 developed an RNA device in S. cerevisiae enabling aptamer-mediated transcript cleavage. This was done by inserting into the 3′ UTR of the transcript of interest a hairpin-shaped formation amenable to cleavage by the ribonuclease Rnt1p and containing an aptamer sequence that leads to inhibited cleavage activity when ligand-bound; in the absence of ligand, Rnt1p cleavage proceeds normally and the transcript, with its polyA tail removed, is quickly degraded.…”

“…Babiskin and Smolke 120 employed three different strategies to tune the monotonically increasing ligand vs gene expression response curve. Changes to a key region of the hairpin sequence controlling cleavage efficiency yielded various combinations of vertical extension and leakage tuning, typically with only slight horizontal scaling .…”

Applications and advances of metabolite biosensors for metabolic engineering

“…Another mechanism involves metabolite-controlled activity of an RNase III enzyme, Rnt1p (Fig. 4d), as demonstrated in the case of a theophylline-responsive biosensor (Babiskin and Smolke, 2011). The binding of theophylline to the aptamer triggers a structural change that inhibits Rnt1p processing, thus increasing the stability of the transcript.…”

Synthetic Biology: Advancing the Design of Diverse Genetic Systems

“…Specific tuning strategies can be established to predictably adjust specific performance measures. For example, the basal activity of an RNA-based I/O device can be efficiently and predictably reduced by implementing multiple copies of the device in tandem (56, 57). As another example, the input sensitivity of a genetic transcriptional device can be improved by introducing device cascading, which increases the degree of cooperativity of the combined device (58).…”