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A synthetic library of RNA control modules for predictable tuning of gene expression in yeast.

Babiskin AH, Smolke CD - Mol. Syst. Biol. (2011)

Bottom Line: Advances in synthetic biology have resulted in the development of genetic tools that support the design of complex biological systems encoding desired functions.This new class of control elements can be combined with any promoter to support titration of regulatory strategies encoded in transcriptional regulators and thus more sophisticated control schemes.We applied these synthetic controllers to the systematic titration of flux through the ergosterol biosynthesis pathway, providing insight into endogenous control strategies and highlighting the utility of this control module library for manipulating and probing biological systems.

View Article: PubMed Central - PubMed

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

ABSTRACT
Advances in synthetic biology have resulted in the development of genetic tools that support the design of complex biological systems encoding desired functions. The majority of efforts have focused on the development of regulatory tools in bacteria, whereas fewer tools exist for the tuning of expression levels in eukaryotic organisms. Here, we describe a novel class of RNA-based control modules that provide predictable tuning of expression levels in the yeast Saccharomyces cerevisiae. A library of synthetic control modules that act through posttranscriptional RNase cleavage mechanisms was generated through an in vivo screen, in which structural engineering methods were applied to enhance the insulation and modularity of the resulting components. This new class of control elements can be combined with any promoter to support titration of regulatory strategies encoded in transcriptional regulators and thus more sophisticated control schemes. We applied these synthetic controllers to the systematic titration of flux through the ergosterol biosynthesis pathway, providing insight into endogenous control strategies and highlighting the utility of this control module library for manipulating and probing biological systems.

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Genetic control elements based on Rnt1p hairpins. (A) Consensus elements of an Rnt1p hairpin. Color scheme is as follows: cleavage efficiency box (CEB), red; binding stability box (BSB), blue; initial binding and positioning box (IBPB), green. Black triangles represent location of cleavage sites. The clamp region is a synthetic sequence that acts to insulate and maintain the structure of the control element. (B) Schematic illustrating the mechanism by which Rnt1p hairpins act as gene control elements when placed in the 3′ UTR of a gene of interest (goi). Barrels represent protein molecules. (C) Sequences and structures of Rnt1p hairpin controls. (D) The transcript and protein levels associated with Rnt1p hairpins and their corresponding mutated tetraloop (CAUC) controls support that the observed gene regulatory activity is due to Rnt1p processing. Normalized protein expression levels are determined by measuring the median GFP levels from a cell population harboring the appropriate construct through flow cytometry analysis and values are reported relative to that from an identical construct lacking a hairpin module (no insert). Reported values and their error are calculated from the mean and standard deviation, respectively, from the three identically grown samples. Transcript levels are determined by measuring transcript levels of yEGFP3 and a housekeeping gene, ACT1, through qRT–PCR and normalizing the yEGFP3 levels with their corresponding ACT1 levels. Normalized transcript levels are reported relative to that from an identical construct lacking a hairpin module. Reported values and their error are calculated from the mean and standard deviation, respectively, from three identically prepared qRT–PCR reactions. Source data is available for this figure at www.nature.com/msb.
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f1: Genetic control elements based on Rnt1p hairpins. (A) Consensus elements of an Rnt1p hairpin. Color scheme is as follows: cleavage efficiency box (CEB), red; binding stability box (BSB), blue; initial binding and positioning box (IBPB), green. Black triangles represent location of cleavage sites. The clamp region is a synthetic sequence that acts to insulate and maintain the structure of the control element. (B) Schematic illustrating the mechanism by which Rnt1p hairpins act as gene control elements when placed in the 3′ UTR of a gene of interest (goi). Barrels represent protein molecules. (C) Sequences and structures of Rnt1p hairpin controls. (D) The transcript and protein levels associated with Rnt1p hairpins and their corresponding mutated tetraloop (CAUC) controls support that the observed gene regulatory activity is due to Rnt1p processing. Normalized protein expression levels are determined by measuring the median GFP levels from a cell population harboring the appropriate construct through flow cytometry analysis and values are reported relative to that from an identical construct lacking a hairpin module (no insert). Reported values and their error are calculated from the mean and standard deviation, respectively, from the three identically grown samples. Transcript levels are determined by measuring transcript levels of yEGFP3 and a housekeeping gene, ACT1, through qRT–PCR and normalizing the yEGFP3 levels with their corresponding ACT1 levels. Normalized transcript levels are reported relative to that from an identical construct lacking a hairpin module. Reported values and their error are calculated from the mean and standard deviation, respectively, from three identically prepared qRT–PCR reactions. Source data is available for this figure at www.nature.com/msb.

Mentions: Rnt1p is an RNase III enzyme that cleaves consensus hairpin structures in S. cerevisiae. For a hairpin to be effectively recognized and cleaved by Rnt1p, it must have the following consensus elements: an AGNN tetraloop and four base pairs immediately below the tetraloop (Figure 1A). An Rnt1p substrate can be divided into three critical regions: the initial binding and positioning box (IBPB), comprising the tetraloop; the binding stability box (BSB), comprising the base-paired region immediately adjacent to the tetraloop; and the cleavage efficiency box (CEB), comprising the region containing and surrounding the cleavage site (Lamontagne et al, 2003). The CEB has no reported sequence or structural requirements. Rnt1p will initially position itself, bind to the tetraloop and cleave the hairpin at two locations within the CEB, that is, between the fourteenth and fifteenth nts upstream of the tetraloop and the sixteenth and seventeenth nts downstream of the tetraloop. Most naturally occurring Rnt1p hairpins have been identified in non-coding RNAs (ncRNAs), in which Rnt1p has a critical role in ncRNA processing (Elela et al, 1996; Chanfreau et al, 1997, 1998). Synthetic trans-acting RNA guide strands were recently used to direct Rnt1p processing of a target ncRNA (Lamontagne and Abou Elela, 2007). Rnt1p hairpins have also been identified within the coding region of at least one endogenous yeast gene, MIG2, in which Rnt1p was shown to have a role in controlling expression levels of that gene (Ge et al, 2005). However, the ability of Rnt1p hairpins to function as genetic control modules in regulating the expression of heterologous genes has not been previously examined.


A synthetic library of RNA control modules for predictable tuning of gene expression in yeast.

Babiskin AH, Smolke CD - Mol. Syst. Biol. (2011)

Genetic control elements based on Rnt1p hairpins. (A) Consensus elements of an Rnt1p hairpin. Color scheme is as follows: cleavage efficiency box (CEB), red; binding stability box (BSB), blue; initial binding and positioning box (IBPB), green. Black triangles represent location of cleavage sites. The clamp region is a synthetic sequence that acts to insulate and maintain the structure of the control element. (B) Schematic illustrating the mechanism by which Rnt1p hairpins act as gene control elements when placed in the 3′ UTR of a gene of interest (goi). Barrels represent protein molecules. (C) Sequences and structures of Rnt1p hairpin controls. (D) The transcript and protein levels associated with Rnt1p hairpins and their corresponding mutated tetraloop (CAUC) controls support that the observed gene regulatory activity is due to Rnt1p processing. Normalized protein expression levels are determined by measuring the median GFP levels from a cell population harboring the appropriate construct through flow cytometry analysis and values are reported relative to that from an identical construct lacking a hairpin module (no insert). Reported values and their error are calculated from the mean and standard deviation, respectively, from the three identically grown samples. Transcript levels are determined by measuring transcript levels of yEGFP3 and a housekeeping gene, ACT1, through qRT–PCR and normalizing the yEGFP3 levels with their corresponding ACT1 levels. Normalized transcript levels are reported relative to that from an identical construct lacking a hairpin module. Reported values and their error are calculated from the mean and standard deviation, respectively, from three identically prepared qRT–PCR reactions. Source data is available for this figure at www.nature.com/msb.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3094065&req=5

f1: Genetic control elements based on Rnt1p hairpins. (A) Consensus elements of an Rnt1p hairpin. Color scheme is as follows: cleavage efficiency box (CEB), red; binding stability box (BSB), blue; initial binding and positioning box (IBPB), green. Black triangles represent location of cleavage sites. The clamp region is a synthetic sequence that acts to insulate and maintain the structure of the control element. (B) Schematic illustrating the mechanism by which Rnt1p hairpins act as gene control elements when placed in the 3′ UTR of a gene of interest (goi). Barrels represent protein molecules. (C) Sequences and structures of Rnt1p hairpin controls. (D) The transcript and protein levels associated with Rnt1p hairpins and their corresponding mutated tetraloop (CAUC) controls support that the observed gene regulatory activity is due to Rnt1p processing. Normalized protein expression levels are determined by measuring the median GFP levels from a cell population harboring the appropriate construct through flow cytometry analysis and values are reported relative to that from an identical construct lacking a hairpin module (no insert). Reported values and their error are calculated from the mean and standard deviation, respectively, from the three identically grown samples. Transcript levels are determined by measuring transcript levels of yEGFP3 and a housekeeping gene, ACT1, through qRT–PCR and normalizing the yEGFP3 levels with their corresponding ACT1 levels. Normalized transcript levels are reported relative to that from an identical construct lacking a hairpin module. Reported values and their error are calculated from the mean and standard deviation, respectively, from three identically prepared qRT–PCR reactions. Source data is available for this figure at www.nature.com/msb.
Mentions: Rnt1p is an RNase III enzyme that cleaves consensus hairpin structures in S. cerevisiae. For a hairpin to be effectively recognized and cleaved by Rnt1p, it must have the following consensus elements: an AGNN tetraloop and four base pairs immediately below the tetraloop (Figure 1A). An Rnt1p substrate can be divided into three critical regions: the initial binding and positioning box (IBPB), comprising the tetraloop; the binding stability box (BSB), comprising the base-paired region immediately adjacent to the tetraloop; and the cleavage efficiency box (CEB), comprising the region containing and surrounding the cleavage site (Lamontagne et al, 2003). The CEB has no reported sequence or structural requirements. Rnt1p will initially position itself, bind to the tetraloop and cleave the hairpin at two locations within the CEB, that is, between the fourteenth and fifteenth nts upstream of the tetraloop and the sixteenth and seventeenth nts downstream of the tetraloop. Most naturally occurring Rnt1p hairpins have been identified in non-coding RNAs (ncRNAs), in which Rnt1p has a critical role in ncRNA processing (Elela et al, 1996; Chanfreau et al, 1997, 1998). Synthetic trans-acting RNA guide strands were recently used to direct Rnt1p processing of a target ncRNA (Lamontagne and Abou Elela, 2007). Rnt1p hairpins have also been identified within the coding region of at least one endogenous yeast gene, MIG2, in which Rnt1p was shown to have a role in controlling expression levels of that gene (Ge et al, 2005). However, the ability of Rnt1p hairpins to function as genetic control modules in regulating the expression of heterologous genes has not been previously examined.

Bottom Line: Advances in synthetic biology have resulted in the development of genetic tools that support the design of complex biological systems encoding desired functions.This new class of control elements can be combined with any promoter to support titration of regulatory strategies encoded in transcriptional regulators and thus more sophisticated control schemes.We applied these synthetic controllers to the systematic titration of flux through the ergosterol biosynthesis pathway, providing insight into endogenous control strategies and highlighting the utility of this control module library for manipulating and probing biological systems.

View Article: PubMed Central - PubMed

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

ABSTRACT
Advances in synthetic biology have resulted in the development of genetic tools that support the design of complex biological systems encoding desired functions. The majority of efforts have focused on the development of regulatory tools in bacteria, whereas fewer tools exist for the tuning of expression levels in eukaryotic organisms. Here, we describe a novel class of RNA-based control modules that provide predictable tuning of expression levels in the yeast Saccharomyces cerevisiae. A library of synthetic control modules that act through posttranscriptional RNase cleavage mechanisms was generated through an in vivo screen, in which structural engineering methods were applied to enhance the insulation and modularity of the resulting components. This new class of control elements can be combined with any promoter to support titration of regulatory strategies encoded in transcriptional regulators and thus more sophisticated control schemes. We applied these synthetic controllers to the systematic titration of flux through the ergosterol biosynthesis pathway, providing insight into endogenous control strategies and highlighting the utility of this control module library for manipulating and probing biological systems.

Show MeSH