title: Thermozymes: Synthetic RNA thermometers based on ribozyme activity.
abstract: Synthetic biology approaches often combine natural building blocks to generate new cellular activities. Here, we make use of two RNA elements to design a regulatory device with novel functionality. The system is based on a hammerhead ribozyme (HHR) that cleaves itself to generate a liberated ribosome-binding site and, thus, permits expression of a downstream gene. We connected a temperature-responsive RNA hairpin to the HHR and, thus, generated a temperature-controlled ribozyme that we call thermozyme. Specifically, a Salmonella RNA thermometer (RNAT) known to modulate small heat shock gene expression by temperature-controlled base-pairing and melting was fused to the ribozyme. Following an in vivo screening approach, we isolated two functional thermozymes. In vivo expression studies and in vitro structure probing experiments support a mechanism in which rising temperatures melt the thermometer structure impairing the self-cleavage reaction of the ribozyme. Since RNA cleavage is necessary to liberate the RBS, these engineered thermozymes shut off gene expression in response to a temperature increase and, thus, act in a reverse manner as the natural RNAT. Our results clearly emphasize the highly modular nature and biotechnological potential of ribozyme-based RNA thermometers.
authors.affiliation: Department of Chemistry and Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany.
authors.forename: Athanasios
authors.initials: A
authors.lastname: Saragliadis
authors.forename: Stefanie S
authors.initials: SS
authors.lastname: Krajewski
authors.forename: Charlotte
authors.initials: C
authors.lastname: Rehm
authors.forename: Franz
authors.initials: F
authors.lastname: Narberhaus
authors.forename: Jörg S
authors.initials: JS
authors.lastname: Hartig
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018869:Heat-Shock Response / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D059367:RNA Cleavage; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
chemical_list: D020121:5' Untranslated Regions; D016337:RNA, Catalytic; C427204:hammerhead ribozyme
keywords: RNA thermometer; gene expression; hammerhead ribozyme; regulatory RNA; riboswitch; temperature