Thermozymes: Synthetic RNA thermometers based on ribozyme activity

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 ribosom...

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Bibliographic Details
Published in:RNA biology 2013-06, Vol.10 (6), p.1009-1016
Main Authors: Saragliadis, Athanasios, Krajewski, Stefanie S., Rehm, Charlotte, Narberhaus, Franz, Hartig, Jörg S.
Format: Article
Language:English
Subjects:
5' Untranslated Regions - genetics
Base Sequence
Binding Sites - genetics
gene expression
hammerhead ribozyme
Heat-Shock Response - genetics
Molecular Sequence Data
Nucleic Acid Conformation
regulatory RNA
Research Paper
Ribosomes - genetics
Ribosomes - metabolism
riboswitch
RNA Cleavage
RNA thermometer
RNA, Catalytic - chemistry
RNA, Catalytic - genetics
RNA, Catalytic - metabolism
Temperature
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Summary: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.
ISSN:1547-6286
1555-8584
DOI:10.4161/rna.24482