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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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Placing an upper limit on the ligand concentration range alters the observed tuning properties.(A) Placing an upper limit on the ligand concentration (L') restricts access to the full response curve. This limit affects the dependence of (B) the dynamic range (η) and (C) the apparent EC50 (EC50APP) on the conformational partitioning constant (K1) and the aptamer association constant (K2) as demonstrated for a thermodynamically-driven riboswitch. The maximum dynamic range (ηmax) is proportional to the difference between regulatory activities for conformations A (KA) and B (KB) normalized to the respective degradation rate constants kdMA and kdMB. (D) Normalized response curves for fixed L' and increasing values of (1+K1)/K2, which equals EC50 under ligand-saturating conditions. Parameter values are identical to those reported in Figure 2 with L' = 60 µM.
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pcbi-1000363-g005: Placing an upper limit on the ligand concentration range alters the observed tuning properties.(A) Placing an upper limit on the ligand concentration (L') restricts access to the full response curve. This limit affects the dependence of (B) the dynamic range (η) and (C) the apparent EC50 (EC50APP) on the conformational partitioning constant (K1) and the aptamer association constant (K2) as demonstrated for a thermodynamically-driven riboswitch. The maximum dynamic range (ηmax) is proportional to the difference between regulatory activities for conformations A (KA) and B (KB) normalized to the respective degradation rate constants kdMA and kdMB. (D) Normalized response curves for fixed L' and increasing values of (1+K1)/K2, which equals EC50 under ligand-saturating conditions. Parameter values are identical to those reported in Figure 2 with L' = 60 µM.

Mentions: In our analyses thus far, we assumed that the maximum ligand concentration always saturates the response curve. However, studies of synthetic riboswitches have demonstrated that the response curve may not be saturated by the accessible upper limit in ligand concentration (Figure 5A) due to various system properties including aptamer affinity, ligand solubility, permeability of the ligand across the cell membrane, and cytotoxicity of the ligand [16], [17], [19], [27]–[29]. Furthermore, natural riboswitches may regularly function in response to physiologically-relevant changes in metabolite concentrations that are much smaller than the ∼1000-fold range necessary to access the full riboswitch response curve. To assess the effect of establishing an upper limit to the ligand concentration, we evaluated the response curve descriptors for a maximum ligand concentration of L'. An apparent EC50 (EC50APP) was calculated according to protein levels at L = 0 and L'.


Design principles for riboswitch function.

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

Placing an upper limit on the ligand concentration range alters the observed tuning properties.(A) Placing an upper limit on the ligand concentration (L') restricts access to the full response curve. This limit affects the dependence of (B) the dynamic range (η) and (C) the apparent EC50 (EC50APP) on the conformational partitioning constant (K1) and the aptamer association constant (K2) as demonstrated for a thermodynamically-driven riboswitch. The maximum dynamic range (ηmax) is proportional to the difference between regulatory activities for conformations A (KA) and B (KB) normalized to the respective degradation rate constants kdMA and kdMB. (D) Normalized response curves for fixed L' and increasing values of (1+K1)/K2, which equals EC50 under ligand-saturating conditions. Parameter values are identical to those reported in Figure 2 with L' = 60 µM.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2666153&req=5

pcbi-1000363-g005: Placing an upper limit on the ligand concentration range alters the observed tuning properties.(A) Placing an upper limit on the ligand concentration (L') restricts access to the full response curve. This limit affects the dependence of (B) the dynamic range (η) and (C) the apparent EC50 (EC50APP) on the conformational partitioning constant (K1) and the aptamer association constant (K2) as demonstrated for a thermodynamically-driven riboswitch. The maximum dynamic range (ηmax) is proportional to the difference between regulatory activities for conformations A (KA) and B (KB) normalized to the respective degradation rate constants kdMA and kdMB. (D) Normalized response curves for fixed L' and increasing values of (1+K1)/K2, which equals EC50 under ligand-saturating conditions. Parameter values are identical to those reported in Figure 2 with L' = 60 µM.
Mentions: In our analyses thus far, we assumed that the maximum ligand concentration always saturates the response curve. However, studies of synthetic riboswitches have demonstrated that the response curve may not be saturated by the accessible upper limit in ligand concentration (Figure 5A) due to various system properties including aptamer affinity, ligand solubility, permeability of the ligand across the cell membrane, and cytotoxicity of the ligand [16], [17], [19], [27]–[29]. Furthermore, natural riboswitches may regularly function in response to physiologically-relevant changes in metabolite concentrations that are much smaller than the ∼1000-fold range necessary to access the full riboswitch response curve. To assess the effect of establishing an upper limit to the ligand concentration, we evaluated the response curve descriptors for a maximum ligand concentration of L'. An apparent EC50 (EC50APP) was calculated according to protein levels at L = 0 and L'.

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