Development of heat-repressible RNA thermosensors in bacteria

The overall goal of this research is to develop small, synthetic, heat-repressible RNA thermosensors. Most RNA thermosensors are heat-inducible, and they function by sequestering the Shine-Dalgarno site in a stem-loop structure at low temperatures and by exposing it to allow expression at high temperatures. Here, we demonstrate the design of heat-repressible thermosensors that display the opposite response to temperature. These thermosensors are located in the 5' UTR upstream of the Shine-Dalgarno site, where they contain a recognition site for ribonuclease E, an enzyme native to Escherichia coli that binds at the recognition site and degrades the mRNA. At low temperatures, the recognition site is sequestered in a stem-loop structure. At high temperatures, the stem-loop unfolds and exposes the recognition site, and thus the mRNA is degraded, turning off gene expression. Synthetic heat-repressible RNA thermosensors were designed and tested in vivo by varying length, number of recognition sites, size of the loop, and predicted melting temperature of the stem. Cells containing a thermosensor-controlled GFP as well as an internal control RFP were grown at a variety of temperatures ranging from 15'C to 37'C, with the best performing thermosensor showing greater than four-fold change in expression from low to high temperatures. These temperature-repressible thermosensors are small, do not require expression of protein regulators, and function independently of chemical inducers that incur additional costs in scale-up. In addition, they are simple to design and can be implemented in the optimization of a range of metabolic processes.