De novo design of heat-repressible RNA thermosensors in E. coli. Issue 12 (15th May 2015)

• Record Type: Journal Article
• Authors: Hoynes-O'Connor, Allison; Hinman, Kristina; Kirchner, Lukas; Moon, Tae Seok
• Is Part Of: Nucleic acids research. Volume 43:Issue 12(2015)
• Page Start: 6166
• Page End: 6179
• Publication Date: 2015-05-15
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkv499
• Languages: English
• ISSNs: 0305-1048

Abstract

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.