An adaptor from translational to transcriptional control enables predictable assembly of complex regulation

Abstract

Bacterial regulators of transcriptional elongation are versatile units for building custom genetic switches, as they control the expression of both coding and noncoding RNAs, act on multigene operons and can be predictably tethered into higher-order regulatory functions (a property called composability). Yet the less versatile bacterial regulators of translational initiation are substantially easier to engineer. To bypass this tradeoff, we have developed an adaptor that converts regulators of translational initiation into regulators of transcriptional elongation in Escherichia coli. We applied this adaptor to the construction of several transcriptional attenuators and activators, including a small molecule-triggered attenuator and a group of five mutually orthogonal riboregulators that we assembled into NOR gates of two, three or four RNA inputs. Continued application of our adaptor should produce large collections of transcriptional regulators whose inherent composability can facilitate the predictable engineering of complex synthetic circuits.

Principles of genetic circuit design

“…RNAP flux can also be altered with invertases that change the orientation of promoters, terminators, or gene sequences. Additionally, RNA translational repressors, such as RNA-IN/OUT, can be converted to control RNAP flux 80,81 . In this section, we describe recent advances in these methods and analyze the impact that each regulator has on circuit response.…”

“…In the natural system, RNA-OUT binds to a specific sequence at the 5′ end of an mRNA (RNA-IN) to occlude ribosome binding and increase mRNA degradation 140–142 . Arkin and co-workers retooled this system to repress transcription, instead of translation, using a transcriptional adaptor from the tna operon 80 . The tna regulatory element is composed of a ribosome binding site (RBS), the coding sequence for a short peptide called tnaC , a Rho binding site and an RNAP pause site that facilitates Rho-mediated transcription termination.…”

Developments in the Tools and Methodologies of Synthetic Biology

“…However, unlike transcriptional circuits, there is no common signal carrier and thus they cannot be as easily composed into complex regulatory designs. By repurposing a regulatory element from the tnaCAB operon of E. coli , Liu et al (2012), have created an adapter to convert translational regulators into transcriptional regulators (Liu et al, 2012). The 5′-end of the operon codes for a short leader peptide, TnaC that stalls the ribosome in the presence of free tryptophan.…”

Regulatory RNAs

“…2 tnaC: is a regulatory element that contains a Ribosomal Binding Site, short leader peptide code and a rho terminator. 1 it is based on the regulatory element from tna operon in E. coli.…”

“…7C). Liu et al 1 developed an adaptor (Fig. 7D) that converts a translational regulator into a transcriptional regulator.…”