Limits...
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.

Show MeSH
In vivo characterization of the selected Rnt1p cleavage library. (A) The gene regulatory range of the Rnt1p library spans a broad range of protein expression levels. (B) The transcript and protein levels associated with all Rnt1p library members and their corresponding mutated tetraloop (CAUC) controls supports that the observed gene regulatory activity is due to Rnt1p processing. (C) Correlation analysis of protein and transcript levels from the Rnt1p hairpin library members supports a strong correlation between the two measures of gene regulatory activity. All normalized protein and transcript levels and their error are determined as described in Figure 1D. Source data is available for this figure at www.nature.com/msb.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3094065&req=5

f3: In vivo characterization of the selected Rnt1p cleavage library. (A) The gene regulatory range of the Rnt1p library spans a broad range of protein expression levels. (B) The transcript and protein levels associated with all Rnt1p library members and their corresponding mutated tetraloop (CAUC) controls supports that the observed gene regulatory activity is due to Rnt1p processing. (C) Correlation analysis of protein and transcript levels from the Rnt1p hairpin library members supports a strong correlation between the two measures of gene regulatory activity. All normalized protein and transcript levels and their error are determined as described in Figure 1D. Source data is available for this figure at www.nature.com/msb.

Mentions: Previous in vitro studies had identified three key regions in Rnt1p hairpins: the cleavage efficiency box (CEB), the binding stability box and the initial binding and positioning box (Lamontagne et al, 2003). The CEB region affects the processing of the hairpin stem by Rnt1p, such that nucleotide (nt) modifications in this region are expected to specifically modulate the cleavage rate. We created an Rnt1p hairpin library by randomizing the CEB region (12 nt). This library was placed downstream of a fluorescent reporter protein and a cell-based screening assay was used to identify functional members of the library that resulted in lowered fluorescence levels. The functional Rnt1p hairpin library comprises 16 unique sequences that span a large gene regulatory range—from 8 to 85% (Figure 3A)—and are fairly evenly distributed across this range. The negative controls for each sequence (constructed by mutating the required consensus tetraloop sequence) demonstrated that the majority of gene knockdown observed from each hairpin is due to Rnt1p processing (Figure 3B). A correlation analysis on the transcript and protein levels for each library hairpin construct indicated a strong positive correlation and a strong preservation of rank order between the two in vivo regulatory measurements (Figure 3C). Characterization of the hairpin library in a different genetic context supported the broader utility of these control modules for providing predictable gene control.


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

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

In vivo characterization of the selected Rnt1p cleavage library. (A) The gene regulatory range of the Rnt1p library spans a broad range of protein expression levels. (B) The transcript and protein levels associated with all Rnt1p library members and their corresponding mutated tetraloop (CAUC) controls supports that the observed gene regulatory activity is due to Rnt1p processing. (C) Correlation analysis of protein and transcript levels from the Rnt1p hairpin library members supports a strong correlation between the two measures of gene regulatory activity. All normalized protein and transcript levels and their error are determined as described in Figure 1D. 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

f3: In vivo characterization of the selected Rnt1p cleavage library. (A) The gene regulatory range of the Rnt1p library spans a broad range of protein expression levels. (B) The transcript and protein levels associated with all Rnt1p library members and their corresponding mutated tetraloop (CAUC) controls supports that the observed gene regulatory activity is due to Rnt1p processing. (C) Correlation analysis of protein and transcript levels from the Rnt1p hairpin library members supports a strong correlation between the two measures of gene regulatory activity. All normalized protein and transcript levels and their error are determined as described in Figure 1D. Source data is available for this figure at www.nature.com/msb.
Mentions: Previous in vitro studies had identified three key regions in Rnt1p hairpins: the cleavage efficiency box (CEB), the binding stability box and the initial binding and positioning box (Lamontagne et al, 2003). The CEB region affects the processing of the hairpin stem by Rnt1p, such that nucleotide (nt) modifications in this region are expected to specifically modulate the cleavage rate. We created an Rnt1p hairpin library by randomizing the CEB region (12 nt). This library was placed downstream of a fluorescent reporter protein and a cell-based screening assay was used to identify functional members of the library that resulted in lowered fluorescence levels. The functional Rnt1p hairpin library comprises 16 unique sequences that span a large gene regulatory range—from 8 to 85% (Figure 3A)—and are fairly evenly distributed across this range. The negative controls for each sequence (constructed by mutating the required consensus tetraloop sequence) demonstrated that the majority of gene knockdown observed from each hairpin is due to Rnt1p processing (Figure 3B). A correlation analysis on the transcript and protein levels for each library hairpin construct indicated a strong positive correlation and a strong preservation of rank order between the two in vivo regulatory measurements (Figure 3C). Characterization of the hairpin library in a different genetic context supported the broader utility of these control modules for providing predictable gene control.

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