Dataset Information

Engineering and characterization of fluorogenic glycine riboswitches.

ABSTRACT: A set of 12 fluorogenic glycine riboswitches with different thermodynamic and kinetic response properties was engineered. For the design of functional riboswitches, a three-part RNA approach was applied based on the idea of linking a RNA sensor, transmitter and actuator part together. For the RNA sensor and actuator part, we used the tandem glycine aptamer structure from Bacillus subtillis, and fluorogenic aptamer Spinach, respectively. To achieve optimal signal transduction from the sensor to the actuator, a riboswitch library with variable transmitter was screened with a microfluidic large-scale integration chip. This allowed us to establish the complete thermodynamic binding profiles of the riboswitch library. Glycine dissociation constants of the 12 strong fluorescence response riboswitches varied between 99.7 and 570 ?M. Furthermore, the kinetic glycine binding (k(on)), and dissociation (k(off)) rates, and corresponding energy barriers of the 10 strongest fluorescence response riboswitches were determined with the same chip platform. k(on) and k(off) were in the order of 10(-3)s(-1) and 10(-2)s(-1), respectively. Conclusively, we demonstrate that systematic screening of synthetic and natural linked RNA parts with microfluidic chip technology is an effective approach to rapidly generate fluorogenic metabolite riboswitches with a broad range of biophysical response properties.

SUBMITTER: Ketterer S

PROVIDER: S-EPMC4937332 | biostudies-other | 2016 Jul

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Engineering and characterization of fluorogenic glycine riboswitches.

Ketterer Simon S Gladis Lukas L Kozica Adnan A Meier Matthias M

Nucleic acids research 20160524 12

A set of 12 fluorogenic glycine riboswitches with different thermodynamic and kinetic response properties was engineered. For the design of functional riboswitches, a three-part RNA approach was applied based on the idea of linking a RNA sensor, transmitter and actuator part together. For the RNA sensor and actuator part, we used the tandem glycine aptamer structure from Bacillus subtillis, and fluorogenic aptamer Spinach, respectively. To achieve optimal signal transduction from the sensor to t ...[more]

PMID: 27220466

Similar Datasets

Project description:We report here the behavior of naturally occurring and rationally engineered preQ1 riboswitches and their application to inducible gene regulation in mycobacteria. Because mycobacteria lack preQ1 biosynthetic genes, we hypothesized that preQ1 could be used as an exogenous nonmetabolite ligand to control riboswitches in mycobacteria. Selected naturally occurring preQ1 riboswitches were assayed and successfully drove preQ1-dependent repression of a green fluorescent protein reporter in Mycobacterium smegmatis Using structure-based design, we engineered three preQ1 riboswitches from Thermoanaerobacter tencongensis, Bacillus subtilis, and Lactobacillus rhamnosus toward achieving higher response ratios and increased repression. Assuming a steady-state model, variants of the T. tencongensis riboswitch most closely followed the predicted trends. Unexpectedly, the preQ1 dose response was best described by a model with a second, independent preQ1 binding site. This behavior was general to the preQ1 riboswitch family, since the wild type and rationally designed mutants of riboswitches from all three bacteria behaved analogously. Across all variants, the response ratios, which describe expression in the absence versus the presence of preQ1, ranged from <2 to ∼10, but repression in all cases was incomplete up to 1 mM preQ1. By reducing the transcript expression level, we obtained a preQ1 riboswitch variant appropriate for inducible knockdown applications. We further showed that the preQ1 response is reversible, is titratable, and can be used to control protein expression in mycobacteria within infected macrophages. By engineering naturally occurring preQ1 riboswitches, we have not only extended the tools available for inducible gene regulation in mycobacteria but also uncovered new behavior of these riboswitches.IMPORTANCE Riboswitches are elements found in noncoding regions of mRNA that regulate gene expression, typically in response to an endogenous metabolite. Riboswitches have emerged as important tools for inducible gene expression in diverse organisms. We noted that mycobacteria lack the biosynthesis genes for preQ1, a ligand for riboswitches from diverse bacteria. Predicting that preQ1 is not present in mycobacteria, we showed that it controls optimized riboswitches appropriate for gene knockdown applications. Further, the riboswitch response is subject to a second independent preQ1 binding event that has not been previously documented. By engineering naturally occurring riboswitches, we have uncovered a new behavior, with implications for riboswitch function in its native context, and extended the tools available for inducible gene regulation in mycobacteria.

Project description:The ability to sense and respond to intracellular metabolite levels enables cells to adapt to environmental conditions. Many prokaryotes use riboswitches-structured RNA elements usually located in the 5' untranslated region of mRNAs-to sense intracellular metabolites and respond by modulating gene expression. The corrinoid riboswitch class, which responds to adenosylcobalamin (coenzyme B12) and related metabolites, is among the most widespread in bacteria. The structural elements for corrinoid binding and the requirement for a kissing loop interaction between the aptamer and expression platform domains have been established for several corrinoid riboswitches. However, the conformational changes in the expression platform that modulate gene expression in response to corrinoid binding remain unknown. Here, we employ an in vivo GFP reporter system in Bacillus subtilis to define alternative secondary structures in the expression platform of a corrinoid riboswitch from Priestia megaterium by disrupting and restoring base-pairing interactions. Moreover, we report the discovery and characterization of the first riboswitch known to activate gene expression in response to corrinoids. In both cases, mutually exclusive RNA secondary structures are responsible for promoting or preventing the formation of an intrinsic transcription terminator in response to the corrinoid binding state of the aptamer domain. Knowledge of these regulatory mechanisms allowed us to develop synthetic corrinoid riboswitches that convert repressing riboswitches to riboswitches that robustly induce gene expression in response to corrinoids. Due to their high expression levels, low background, and over 100-fold level of induction, these synthetic riboswitches have potential use as biosensors or genetic tools. IMPORTANCE In addition to proteins, microbes can use structured RNAs such as riboswitches for the important task of regulating gene expression. Riboswitches control gene expression by changing their structure in response to binding a small molecule and are widespread among bacteria. Here we determine the mechanism of regulation in a riboswitch that responds to corrinoids-a family of coenzymes related to vitamin B12. We report the alternative RNA secondary structures that couple corrinoid sensing with response in a repressing and novel activating corrinoid riboswitch. We then applied this knowledge to flipping the regulatory sign by constructing synthetic riboswitches that activate expression to a higher level than the natural one. In the process, we observed patterns in which sequence, in addition to structure, impacts function in paired RNA regions. The synthetic riboswitches we describe here have potential applications as biosensors.

Dataset Information

Engineering and characterization of fluorogenic glycine riboswitches.

Publications

Engineering and characterization of fluorogenic glycine riboswitches.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets