De novodesign of heat-repressible RNA thermosensors inE. coli

Abstract

RNA-based temperature sensing is common in bacteria that live in fluctuating environments. Most naturally-occurring RNA thermosensors are heat-inducible, have long sequences, and function by sequestering the ribosome binding site in a hairpin structure at lower temperatures. Here, we demonstrate the de novo design of short, heat-repressible RNA thermosensors. These thermosensors contain a cleavage site for RNase E, an enzyme native to Escherichia coli and many other organisms, in the 5′ untranslated region of the target gene. At low temperatures, the cleavage site is sequestered in a stem–loop, and gene expression is unobstructed. At high temperatures, the stem–loop unfolds, allowing for mRNA degradation and turning off expression. We demonstrated that these thermosensors respond specifically to temperature and provided experimental support for the central role of RNase E in the mechanism. We also demonstrated the modularity of these RNA thermosensors by constructing a three-input composite circuit that utilizes transcriptional, post-transcriptional, and post-translational regulation. A thorough analysis of the 24 thermosensors allowed for the development of design guidelines for systematic construction of similar thermosensors in future applications. These short, modular RNA thermosensors can be applied to the construction of complex genetic circuits, facilitating rational reprogramming of cellular processes for synthetic biology applications.

Applications and limitations of regulatoryRNAelements in synthetic biology and biotechnology

“…) and GFP (Hoynes‐O'Connor et al . ). Moreover, RNATs are not necessarily limited to translational regulation, as at least one group has successfully exploited RNAT structures to create regulatory elements that control transcription termination in a temperature‐dependent manner (Rossmanith et al .…”

“…; Berens and Suess ; Hoynes‐O'Connor et al . ; Rodrigues and Rodrigues ). This section will discuss a few of the more interesting applications of synthetic riboswitches that have been tested in E. coli , as well as their implications.…”

Regulatory effects of cotranscriptional RNA structure formation and transitions

“…Thermal triggers and physiochemical conditions in vivo also have significant effects on RNA folding pathways; for example, RNA thermosensors (RNATs) can regulate gene expression during cold and shock response in response to fluctuations in temperature, magnesium ion (Mg 2+ ) concentration, and pH . These RNATs possess a RNase E cleavage site, an enzyme found in E. coli and plenty of other organisms, in the 5′‐UTR of the target gene.…”

“…The cleavage site is sequestered in a stem‐loop at low temperatures, thereby gene expression is not being blocked. Whereas, the stem‐loop unfolds at high temperatures, permitting mRNA degradation and turning off expression of genes . The stability of a compact RNA structure is exquisitely sensitive to the types and concentrations of ion that are present in vivo , especially Mg 2+ .…”

Design of a Toolbox of RNA Thermometers

“…The temperature response of these thermometers was designed on the basis of the melting temperature of the minimum free energy structure, with an increased stem length, a smaller hairpin loop, or a reduction in number of bulges resulting in an increase in the melting temperature [9,10]. More recently, RNA thermometers have also been designed to be heat-repressible using an RNase E-mediated mechanism [4]. These previous studies have provided important results towards an elucidation of mechanisms underlying RNA thermometer operation, modulation of their response to temperature, and towards prediction of their behaviour.…”