Abstract
RNAs play major roles in the regulation of gene expression. Hence, designer RNA molecules are increasingly explored as regulatory switches in synthetic biology. Among these, the TetR-binding RNA aptamer was selected by its ability to compete with operator DNA for binding to the bacterial repressor TetR. A fortuitous finding was that induction of TetR by tetracycline abolishes both RNA aptamer and operator DNA binding in TetR. This enabled numerous applications exploiting both the specificity of the RNA aptamer and the efficient gene repressor properties of TetR. Here, we present the crystal structure of the TetR-RNA aptamer complex at 2.7 Å resolution together with a comprehensive characterization of the TetR–RNA aptamer versus TetR–operator DNA interaction using site-directed mutagenesis, size exclusion chromatography, electrophoretic mobility shift assays and isothermal titration calorimetry. The fold of the RNA aptamer bears no resemblance to regular B-DNA, and neither does the thermodynamic characterization of the complex formation reaction. Nevertheless, the functional aptamer-binding epitope of TetR is fully contained within its DNA-binding epitope. In the RNA aptamer complex, TetR adopts the well-characterized DNA-binding-competent conformation of TetR, thus revealing how the synthetic TetR-binding aptamer strikes the chords of the bimodal allosteric behaviour of TetR to function as a synthetic regulator.
Highlights
Short and long non-coding RNAs are important regulators of gene expression in all kingdoms of life
RNA molecules have become prominent in synthetic biology, and small regulatory RNAs, synthetic riboswitches and allosterically controlled ribozymes are being investigated as regulatory devices in the design of genetic circuits and networks [1]
RNA-based sensor domains that bind their target with extraordinary high affinity and specificity can be identified de novo by in vitro selection (SELEX, Systematic Evolution of Ligands by Exponential enrichment) [4,5]
Summary
Short and long non-coding RNAs are important regulators of gene expression in all kingdoms of life. RNA molecules have become prominent in synthetic biology, and small regulatory RNAs, synthetic riboswitches and allosterically controlled ribozymes are being investigated as regulatory devices in the design of genetic circuits and networks [1] Rapid progress in this development resulted in a swift transition from simple proof of concept to sophisticated applications targeting complex problems [2,3]. RNA-based sensor domains that bind their target with extraordinary high affinity and specificity can be identified de novo by in vitro selection (SELEX, Systematic Evolution of Ligands by Exponential enrichment) [4,5]. These so called aptamers can adopt defined three-dimensional structures such as binding pockets or cleft-like interaction surfaces similar to those found in antibodies [6–9]. One interesting example is an RNA aptamer that is able to block operator binding in the bacterial transcription regulator TetR [10]
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