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Design principles for riboswitch function.

Beisel CL, Smolke CD - PLoS Comput. Biol. (2009)

Bottom Line: We also found that practical system restrictions, such as an upper limit on ligand concentration, can significantly alter the requirements for riboswitch performance, necessitating alternative tuning strategies.From our results, we developed a set of general design principles for synthetic riboswitches.Our results also provide a foundation from which to investigate how natural riboswitches are tuned to meet systems-level regulatory demands.

View Article: PubMed Central - PubMed

Affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA.

ABSTRACT
Scientific and technological advances that enable the tuning of integrated regulatory components to match network and system requirements are critical to reliably control the function of biological systems. RNA provides a promising building block for the construction of tunable regulatory components based on its rich regulatory capacity and our current understanding of the sequence-function relationship. One prominent example of RNA-based regulatory components is riboswitches, genetic elements that mediate ligand control of gene expression through diverse regulatory mechanisms. While characterization of natural and synthetic riboswitches has revealed that riboswitch function can be modulated through sequence alteration, no quantitative frameworks exist to investigate or guide riboswitch tuning. Here, we combined mathematical modeling and experimental approaches to investigate the relationship between riboswitch function and performance. Model results demonstrated that the competition between reversible and irreversible rate constants dictates performance for different regulatory mechanisms. We also found that practical system restrictions, such as an upper limit on ligand concentration, can significantly alter the requirements for riboswitch performance, necessitating alternative tuning strategies. Previous experimental data for natural and synthetic riboswitches as well as experiments conducted in this work support model predictions. From our results, we developed a set of general design principles for synthetic riboswitches. Our results also provide a foundation from which to investigate how natural riboswitches are tuned to meet systems-level regulatory demands.

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Mutational analysis of a synthetic riboswitch supports model predictions.(A) Mutations made to the aptamer stem of the parent synthetic riboswitch (m1–4) are anticipated to solely modulate conformational partitioning (K1). The theophylline-responsive riboswitch controls Tn10-β-Galactosidase levels through RBS sequestration, thereby repressing translation. Mutations were also introduced to lock the riboswitch in either conformation A (mA) or conformation B (mB). The RBS and start codon are highlighted in orange and green, respectively. (B) β-Galactosidase assay results are reported in Miller Units (MU) for each riboswitch variant in the presence (•) or absence (○) of 1 mM theophylline. Dynamic range (η) is calculated as the difference between high and low expression levels, where all values were below the theoretical maximum of 15,600 MU as determined by the difference between mB with theophylline and mA without theophylline. The positive control construct (empty) harbors only the RBS and aptamer basal stem. A slight increase in β-Galactosidase activity was observed in the presence of theophylline for the control construct. (C,D) Theophylline response curves for riboswitch variants: parent (yellow), m1 (red), m2 (orange), m3 (green), and m4 (blue). (C) Raw data and (D) normalized data illustrate the predicted shift in both basal levels and EC50 for increasing stabilization of conformation B. Data represent independent measurements of triplicate samples, where the standard error was below 5% of each mean value.
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pcbi-1000363-g006: Mutational analysis of a synthetic riboswitch supports model predictions.(A) Mutations made to the aptamer stem of the parent synthetic riboswitch (m1–4) are anticipated to solely modulate conformational partitioning (K1). The theophylline-responsive riboswitch controls Tn10-β-Galactosidase levels through RBS sequestration, thereby repressing translation. Mutations were also introduced to lock the riboswitch in either conformation A (mA) or conformation B (mB). The RBS and start codon are highlighted in orange and green, respectively. (B) β-Galactosidase assay results are reported in Miller Units (MU) for each riboswitch variant in the presence (•) or absence (○) of 1 mM theophylline. Dynamic range (η) is calculated as the difference between high and low expression levels, where all values were below the theoretical maximum of 15,600 MU as determined by the difference between mB with theophylline and mA without theophylline. The positive control construct (empty) harbors only the RBS and aptamer basal stem. A slight increase in β-Galactosidase activity was observed in the presence of theophylline for the control construct. (C,D) Theophylline response curves for riboswitch variants: parent (yellow), m1 (red), m2 (orange), m3 (green), and m4 (blue). (C) Raw data and (D) normalized data illustrate the predicted shift in both basal levels and EC50 for increasing stabilization of conformation B. Data represent independent measurements of triplicate samples, where the standard error was below 5% of each mean value.

Mentions: To begin evaluating how the predicted tuning trends apply to both natural and synthetic riboswitches, we physically manipulated a recently-described synthetic riboswitch functioning through translational repression that up-regulates gene expression (ON behavior) in the presence of theophylline [18] (Figure 6A). Under the naming convention from Figure 1B, conformation A comprises a base-paired structure between the aptamer and RBS, while conformation B includes a formed aptamer and a single-stranded RBS. This riboswitch was selected because it closely resembles natural riboswitches functioning through translational repression, experimental data suggest that this riboswitch operates in the thermodynamically-driven regime [18], the ligand concentration upper limit does not saturate the response curve [28], and the demonstration that different sequences yield different response curves suggests riboswitch tuning [18]. A theophylline concentration of 1 mM was used as an upper limit, as exceeding this concentration inhibited cell growth. In studies performed by Lynch and coworkers, sequences associated with desirable response curves were identified by randomization of the RBS and screening for variants with low basal activity and a large activity increase in the presence of theophylline. Since the randomized sequence was located in a region responsible for conformational partitioning and translation, mutations most likely reflect simultaneous modulation of KA, KB, and K1. We therefore sought to introduce directed mutations to solely modulate individual model parameters and test model predictions for a thermodynamically-driven riboswitch with a ligand concentration upper limit that prevents response curve saturation.


Design principles for riboswitch function.

Beisel CL, Smolke CD - PLoS Comput. Biol. (2009)

Mutational analysis of a synthetic riboswitch supports model predictions.(A) Mutations made to the aptamer stem of the parent synthetic riboswitch (m1–4) are anticipated to solely modulate conformational partitioning (K1). The theophylline-responsive riboswitch controls Tn10-β-Galactosidase levels through RBS sequestration, thereby repressing translation. Mutations were also introduced to lock the riboswitch in either conformation A (mA) or conformation B (mB). The RBS and start codon are highlighted in orange and green, respectively. (B) β-Galactosidase assay results are reported in Miller Units (MU) for each riboswitch variant in the presence (•) or absence (○) of 1 mM theophylline. Dynamic range (η) is calculated as the difference between high and low expression levels, where all values were below the theoretical maximum of 15,600 MU as determined by the difference between mB with theophylline and mA without theophylline. The positive control construct (empty) harbors only the RBS and aptamer basal stem. A slight increase in β-Galactosidase activity was observed in the presence of theophylline for the control construct. (C,D) Theophylline response curves for riboswitch variants: parent (yellow), m1 (red), m2 (orange), m3 (green), and m4 (blue). (C) Raw data and (D) normalized data illustrate the predicted shift in both basal levels and EC50 for increasing stabilization of conformation B. Data represent independent measurements of triplicate samples, where the standard error was below 5% of each mean value.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2666153&req=5

pcbi-1000363-g006: Mutational analysis of a synthetic riboswitch supports model predictions.(A) Mutations made to the aptamer stem of the parent synthetic riboswitch (m1–4) are anticipated to solely modulate conformational partitioning (K1). The theophylline-responsive riboswitch controls Tn10-β-Galactosidase levels through RBS sequestration, thereby repressing translation. Mutations were also introduced to lock the riboswitch in either conformation A (mA) or conformation B (mB). The RBS and start codon are highlighted in orange and green, respectively. (B) β-Galactosidase assay results are reported in Miller Units (MU) for each riboswitch variant in the presence (•) or absence (○) of 1 mM theophylline. Dynamic range (η) is calculated as the difference between high and low expression levels, where all values were below the theoretical maximum of 15,600 MU as determined by the difference between mB with theophylline and mA without theophylline. The positive control construct (empty) harbors only the RBS and aptamer basal stem. A slight increase in β-Galactosidase activity was observed in the presence of theophylline for the control construct. (C,D) Theophylline response curves for riboswitch variants: parent (yellow), m1 (red), m2 (orange), m3 (green), and m4 (blue). (C) Raw data and (D) normalized data illustrate the predicted shift in both basal levels and EC50 for increasing stabilization of conformation B. Data represent independent measurements of triplicate samples, where the standard error was below 5% of each mean value.
Mentions: To begin evaluating how the predicted tuning trends apply to both natural and synthetic riboswitches, we physically manipulated a recently-described synthetic riboswitch functioning through translational repression that up-regulates gene expression (ON behavior) in the presence of theophylline [18] (Figure 6A). Under the naming convention from Figure 1B, conformation A comprises a base-paired structure between the aptamer and RBS, while conformation B includes a formed aptamer and a single-stranded RBS. This riboswitch was selected because it closely resembles natural riboswitches functioning through translational repression, experimental data suggest that this riboswitch operates in the thermodynamically-driven regime [18], the ligand concentration upper limit does not saturate the response curve [28], and the demonstration that different sequences yield different response curves suggests riboswitch tuning [18]. A theophylline concentration of 1 mM was used as an upper limit, as exceeding this concentration inhibited cell growth. In studies performed by Lynch and coworkers, sequences associated with desirable response curves were identified by randomization of the RBS and screening for variants with low basal activity and a large activity increase in the presence of theophylline. Since the randomized sequence was located in a region responsible for conformational partitioning and translation, mutations most likely reflect simultaneous modulation of KA, KB, and K1. We therefore sought to introduce directed mutations to solely modulate individual model parameters and test model predictions for a thermodynamically-driven riboswitch with a ligand concentration upper limit that prevents response curve saturation.

Bottom Line: We also found that practical system restrictions, such as an upper limit on ligand concentration, can significantly alter the requirements for riboswitch performance, necessitating alternative tuning strategies.From our results, we developed a set of general design principles for synthetic riboswitches.Our results also provide a foundation from which to investigate how natural riboswitches are tuned to meet systems-level regulatory demands.

View Article: PubMed Central - PubMed

Affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA.

ABSTRACT
Scientific and technological advances that enable the tuning of integrated regulatory components to match network and system requirements are critical to reliably control the function of biological systems. RNA provides a promising building block for the construction of tunable regulatory components based on its rich regulatory capacity and our current understanding of the sequence-function relationship. One prominent example of RNA-based regulatory components is riboswitches, genetic elements that mediate ligand control of gene expression through diverse regulatory mechanisms. While characterization of natural and synthetic riboswitches has revealed that riboswitch function can be modulated through sequence alteration, no quantitative frameworks exist to investigate or guide riboswitch tuning. Here, we combined mathematical modeling and experimental approaches to investigate the relationship between riboswitch function and performance. Model results demonstrated that the competition between reversible and irreversible rate constants dictates performance for different regulatory mechanisms. We also found that practical system restrictions, such as an upper limit on ligand concentration, can significantly alter the requirements for riboswitch performance, necessitating alternative tuning strategies. Previous experimental data for natural and synthetic riboswitches as well as experiments conducted in this work support model predictions. From our results, we developed a set of general design principles for synthetic riboswitches. Our results also provide a foundation from which to investigate how natural riboswitches are tuned to meet systems-level regulatory demands.

Show MeSH
Related in: MedlinePlus