Rationalizing Context-Dependent Performance of Dynamic RNA Regulatory Devices

The ability of RNA to sense, regulate, and store information is an attractive attribute for a variety of functional applications including the development of regulatory control devices for synthetic biology. RNA folding and function is known to be highly context sensitive, which limits the modularity and reuse of RNA regulatory devices to control different heterologous sequences and genes. We explored the cause and effect of sequence context sensitivity for translational ON riboswitches located in the 5′ UTR, by constructing and screening a library of N-terminal synonymous codon variants. By altering the N-terminal codon usage we were able to obtain RNA devices with a broad range of functional performance properties (ON, OFF, fold-change). Linear regression and calculated metrics were used to rationalize the major determining features leading to optimal riboswitch performance, and to identify multiple interactions between the explanatory metrics. Finally, partial least squared (PLS) analysis was employed in order to understand the metrics and their respective effect on performance. This PLS model was shown to provide good explanation of our library. This study provides a novel multivariant analysis framework to rationalize the codon context performance of allosteric RNA-devices. The framework will also serve as a platform for future riboswitch context engineering endeavors

Rationalizing Context-Dependent Performance of Dynamic RNA Regulatory Devices

The ability of RNA to sense, regulate, and store information is an attractive attribute for a variety of functional applications including the development of regulatory control devices for synthetic biology. RNA folding and function is known to be highly context sensitive, which limits the modularity and reuse of RNA regulatory devices to control different heterologous sequences and genes. We explored the cause and effect of sequence context sensitivity for translational ON riboswitches located in the 5′ UTR, by constructing and screening a library of N-terminal synonymous codon variants. By altering the N-terminal codon usage we were able to obtain RNA devices with a broad range of functional performance properties (ON, OFF, fold-change). Linear regression and calculated metrics were used to rationalize the major determining features leading to optimal riboswitch performance, and to identify multiple interactions between the explanatory metrics. Finally, partial least squared (PLS) analysis was employed in order to understand the metrics and their respective effect on performance. This PLS model was shown to provide good explanation of our library. This study provides a novel multivariant analysis framework to rationalize the codon context performance of allosteric RNA-devices. The framework will also serve as a platform for future riboswitch context engineering endeavors

List of node types and their properties.

<p>List of node types and their properties.</p

List of relationships between nodes.

<p>List of relationships between nodes.</p

SBOL Visual aids rapid communication of variant synthetic DNA designs: multi-color reporter variants.

<p>In this figure we present an example genetic reporter device [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002310#pbio.1002310.ref017" target="_blank">17</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002310#pbio.1002310.ref021" target="_blank">21</a>] to show how a common symbology can highlight the differences between a series of related DNA constructs. (A) The three-color, green fluorescent protein (GFP)-based genetic reporter was designed for easy swapping of each cassette’s promoter by methods based on restriction cloning or recombination, allowing it to be modified to measure promoters for analyzing the effects of growth condition on circuit performance variation (figure modified from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002310#pbio.1002310.ref020" target="_blank">20</a>]). The SBOL Visual symbology (right) highlights that the promoters are variable, and the coloring of the CDS (which is not constrained by SBOL Visual) is used to keep track of which genetic reporter is at which position, including three fluorescent proteins (CFP: cyan, YFP: yellow, RFP: red). Equivalent means for distinguishing sequences include fill/hatch patterns (e.g., for black-and-white publication) or textual labels above the components. (B) The three-color reporter was modified to make a protein fusion between the Yellow Fluorescent Protein gene and the cI repressor from phage lambda (modified from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002310#pbio.1002310.ref021" target="_blank">21</a>] gray and yellow in the SBOL Visual diagram). This shows how the user-specified coloring can add new information to the basic glyphs. This design also swapped around the positions of the different reporter genes, as well as replacing the promoters, to create a regulatory circuit in which the regulatory protein could be directly observed via the protein fusion with YFP. (C) The three-color reporter was modified to a two-color system, and the YFP gene was replaced with a GFP variant, to optimize two-color measurement. This system was used to systematically measure all combinations of 114 promoters and 111 ribosome binding sites (modified from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002310#pbio.1002310.ref018" target="_blank">18</a>]). (D) The DNA sequence of the three-color reporter construct presented in [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002310#pbio.1002310.ref017" target="_blank">17</a>]. Here the promoter and terminator sequences are underlined, and the three color fluorescent genes are indicated by highlighting the text as cyan, yellow, or red. The purple sequences denote ribosome binding sites. This highlights how the SBOL Visual notation in (A) is much easier to quickly understand than the raw sequence, while still clearly communicating the organization of genetic parts.</p

Integration of proteome and metabolic control to show regulation of carbon and nitrogen metabolic fluxes at the protein (enzyme) level

Shown here are the coupling of carbon and nitrogen fluxes at the level of glutamate dehydrogenase (Gdh1p, Gdh2p) and glutamine synthetase (Gln1p), the regulation of arginine biosynthesis at the carbamoyl phosphate synthetase (Cpa1p, Cpa2p) level and amino-acid biosynthesis, and amino-acid sensing by TOR. Selected proteins with levels consistently upregulated (red) with growth independently of culture conditions are shown. Enzymes responsible for the cytosolic 2-oxoglutarate pool: Aco1p and Aco2p, aconitase and putative aconitase isoenzyme; Odc1p and Odc2p, mitochondrial 2-oxoglutarate transporters; Idp2p, NADP-specific isocitrate dehydrogenase. Enzyme subunits coupling the oxidation of succinate to the transfer of electrons to ubiquinone: Sdh1p and Sdh2p, succinate dehydrogenase, flavoprotein, and iron-sulfur protein subunits, respectively. Metabolic diagram from [42, 91, 92] and drawn using Cell Designer [136] and Adobe Illustrator [137].<p><b>Copyright information:</b></p><p>Taken from "Growth control of the eukaryote cell: a systems biology study in yeast"</p><p>http://jbiol.com/content/6/2/4</p><p>Journal of Biology 2007;6(2):4-4.</p><p>Published online 30 Apr 2007</p><p>PMCID:PMC2373899.</p><p></p

Integration of proteome and metabolic control to show regulation of sulfur and C1 (folate) metabolic fluxes at the protein (enzyme) level

Selected proteins with levels consistently upregulated (red) or downregulated (green) with growth independently of culture conditions are shown. Sulfur, C1 metabolism, methyl cycle, methionine and -adenosylmethionine (SAM) fluxes towards methylation of proteins, rRNAs and tRNAs, and protein biosynthesis are shown here. Metabolic pathways and enzymes are from [42,82, 103-105] and the diagram is drawn with Cell Designer [136] and Adobe Illustrator [137]. Reverse methionine biosynthetic pathways [83] have been omitted for clarity. Metabolite abbreviations: THF, tetrahydrofolate; METTHF, 5,10-methylenetetrahydrofolate; MTHPTGLUT, 5-methyltetrahydropteroyltriglutamate (donor of the terminal methyl group in methionine biosynthesis); GT, glutathione; CYS, cysteine; CT, cystathionine; OAHS, -acetylhomoserine; HCYS, homocysteine; MET, methionine; SAM, -adenosylmethionine; SAH, -adenosylhomocysteine; D-SAM, decarboxylated -adenosylmethionine; MTA, methylthioadenosine. Metabolic steps (genes/enzymes): Met10p, sulfite reductase alpha subunit; Ecm17p, sulfite reductase beta subunit; , folylpolyglutamate synthetase (Met7p not detected; the relevance of polyglutamylation in the C1 metabolism branch was demonstrated at the transcriptional level (see text)); Met13p, methylenetetrahydrofolate reductase isozyme; Met6p, methionine synthase; Mes1p, methionyl-tRNA synthetase; Sam1p, S-adenosylmethionine synthetase isozyme; Sam2p, S-adenosylmethionine synthetase isozyme. Sah1p, S-adenosyl-L-homocysteine hydrolase; Ado1p, adenosine kinase.<p><b>Copyright information:</b></p><p>Taken from "Growth control of the eukaryote cell: a systems biology study in yeast"</p><p>http://jbiol.com/content/6/2/4</p><p>Journal of Biology 2007;6(2):4-4.</p><p>Published online 30 Apr 2007</p><p>PMCID:PMC2373899.</p><p></p

Eagle-i record-level citation widget.

<p>Eagle-i record-level citation widget.</p

Record-level versioning and release-level versioning.

<p>Record-level versioning and release-level versioning.</p

Anatomy of a web-based identifier.

<p>An example of an exemplary unique resource identifier (URI) is below; it is comprised of American Standard Code for Information Interchange (ASCII) characters and follows a pattern that starts with a fixed set of characters (URI pattern). That URI pattern is followed by a local identifier (local ID)—an identifier which, by itself, is only guaranteed to be locally unique within the database or source. A local ID is sometimes referred to as an “accession.” Note this figure illustrates the simplest representation; nuances regarding versioning are covered in Lesson 6 and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001414#pbio.2001414.g005" target="_blank">Fig 5</a>.</p