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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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Rate competition dictates riboswitch performance.The relative values of the reversible and irreversible rate constants generally establish three operating regimes: thermodynamically-driven (▪) when reversible rate constants dominate, kinetically-driven (▪) when the rate constants are balanced, and non-functional (□) when irreversible rate constants dominate. Regimes are qualitatively marked for dynamic range and basal and ligand-saturating levels according the ratio of the rate constants for terminator stem formation (kM) and the progression from conformation A to conformation B (k1). Effect of varying kM on (A) dynamic range, (B) basal protein levels and ligand-saturating protein levels, and (C) EC50 for riboswitches functioning through transcriptional termination. In (B), colored pairs show basal (light) and ligand-saturating (dark) protein levels for complete (red), balanced (black), and negligible (blue) transcriptional folding into conformation B. Parameter values for all curves in (A) and (B) and the red curve in (C): k1 = 10−1/s;k1′ = 10/s; k2 = 106/M·s; k2′ = 10−1/s; KA = kP·kMA/kM = 10−3/s; KB = kP·kMB/kM = 10−2/s; kf = 10−11 M/s; kdP = 10−3/s.
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pcbi-1000363-g003: Rate competition dictates riboswitch performance.The relative values of the reversible and irreversible rate constants generally establish three operating regimes: thermodynamically-driven (▪) when reversible rate constants dominate, kinetically-driven (▪) when the rate constants are balanced, and non-functional (□) when irreversible rate constants dominate. Regimes are qualitatively marked for dynamic range and basal and ligand-saturating levels according the ratio of the rate constants for terminator stem formation (kM) and the progression from conformation A to conformation B (k1). Effect of varying kM on (A) dynamic range, (B) basal protein levels and ligand-saturating protein levels, and (C) EC50 for riboswitches functioning through transcriptional termination. In (B), colored pairs show basal (light) and ligand-saturating (dark) protein levels for complete (red), balanced (black), and negligible (blue) transcriptional folding into conformation B. Parameter values for all curves in (A) and (B) and the red curve in (C): k1 = 10−1/s;k1′ = 10/s; k2 = 106/M·s; k2′ = 10−1/s; KA = kP·kMA/kM = 10−3/s; KB = kP·kMB/kM = 10−2/s; kf = 10−11 M/s; kdP = 10−3/s.

Mentions: The second regime begins when either of the irreversible rate constants balances the associated reversible rate constant (either γ1 or γ2 is between zero and one). We call this regime the ‘kinetically-driven’ regime in accord with uses of this term in the study of natural riboswitches [21],[22], where performance is driven by kinetics over energetics. In this regime, riboswitch molecules have fewer opportunities to sample both conformations and bind and release ligand before the irreversible event occurs, where the number of opportunities is governed by the competition between reversible and irreversible rate constants. Since γ1 is coupled to K1 and kfA while γ2 is coupled to K2, both γ1 and γ2 are anticipated to have a significant impact on the response curve and impart several tuning properties distinct to this regime. We initially use riboswitches functioning through transcriptional termination to highlight two of these tuning properties (Figure 3A–C).


Design principles for riboswitch function.

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

Rate competition dictates riboswitch performance.The relative values of the reversible and irreversible rate constants generally establish three operating regimes: thermodynamically-driven (▪) when reversible rate constants dominate, kinetically-driven (▪) when the rate constants are balanced, and non-functional (□) when irreversible rate constants dominate. Regimes are qualitatively marked for dynamic range and basal and ligand-saturating levels according the ratio of the rate constants for terminator stem formation (kM) and the progression from conformation A to conformation B (k1). Effect of varying kM on (A) dynamic range, (B) basal protein levels and ligand-saturating protein levels, and (C) EC50 for riboswitches functioning through transcriptional termination. In (B), colored pairs show basal (light) and ligand-saturating (dark) protein levels for complete (red), balanced (black), and negligible (blue) transcriptional folding into conformation B. Parameter values for all curves in (A) and (B) and the red curve in (C): k1 = 10−1/s;k1′ = 10/s; k2 = 106/M·s; k2′ = 10−1/s; KA = kP·kMA/kM = 10−3/s; KB = kP·kMB/kM = 10−2/s; kf = 10−11 M/s; kdP = 10−3/s.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2666153&req=5

pcbi-1000363-g003: Rate competition dictates riboswitch performance.The relative values of the reversible and irreversible rate constants generally establish three operating regimes: thermodynamically-driven (▪) when reversible rate constants dominate, kinetically-driven (▪) when the rate constants are balanced, and non-functional (□) when irreversible rate constants dominate. Regimes are qualitatively marked for dynamic range and basal and ligand-saturating levels according the ratio of the rate constants for terminator stem formation (kM) and the progression from conformation A to conformation B (k1). Effect of varying kM on (A) dynamic range, (B) basal protein levels and ligand-saturating protein levels, and (C) EC50 for riboswitches functioning through transcriptional termination. In (B), colored pairs show basal (light) and ligand-saturating (dark) protein levels for complete (red), balanced (black), and negligible (blue) transcriptional folding into conformation B. Parameter values for all curves in (A) and (B) and the red curve in (C): k1 = 10−1/s;k1′ = 10/s; k2 = 106/M·s; k2′ = 10−1/s; KA = kP·kMA/kM = 10−3/s; KB = kP·kMB/kM = 10−2/s; kf = 10−11 M/s; kdP = 10−3/s.
Mentions: The second regime begins when either of the irreversible rate constants balances the associated reversible rate constant (either γ1 or γ2 is between zero and one). We call this regime the ‘kinetically-driven’ regime in accord with uses of this term in the study of natural riboswitches [21],[22], where performance is driven by kinetics over energetics. In this regime, riboswitch molecules have fewer opportunities to sample both conformations and bind and release ligand before the irreversible event occurs, where the number of opportunities is governed by the competition between reversible and irreversible rate constants. Since γ1 is coupled to K1 and kfA while γ2 is coupled to K2, both γ1 and γ2 are anticipated to have a significant impact on the response curve and impart several tuning properties distinct to this regime. We initially use riboswitches functioning through transcriptional termination to highlight two of these tuning properties (Figure 3A–C).

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