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Accurate, model-based tuning of synthetic gene expression using introns in S. cerevisiae.

ABSTRACT: Introns are key regulators of eukaryotic gene expression and present a potentially powerful tool for the design of synthetic eukaryotic gene expression systems. However, intronic control over gene expression is governed by a multitude of complex, incompletely understood, regulatory mechanisms. Despite this lack of detailed mechanistic understanding, here we show how a relatively simple model enables accurate and predictable tuning of synthetic gene expression system in yeast using several predictive intron features such as transcript folding and sequence motifs. Using only natural Saccharomyces cerevisiae introns as regulators, we demonstrate fine and accurate control over gene expression spanning a 100 fold expression range. These results broaden the engineering toolbox of synthetic gene expression systems and provide a framework in which precise and robust tuning of gene expression is accomplished.

SUBMITTER: Yofe I

PROVIDER: S-EPMC4072511 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Accurate, model-based tuning of synthetic gene expression using introns in S. cerevisiae.

Yofe Ido I Zafrir Zohar Z Blau Rachel R Schuldiner Maya M Tuller Tamir T Shapiro Ehud E Ben-Yehezkel Tuval T

PLoS genetics 20140626 6

Introns are key regulators of eukaryotic gene expression and present a potentially powerful tool for the design of synthetic eukaryotic gene expression systems. However, intronic control over gene expression is governed by a multitude of complex, incompletely understood, regulatory mechanisms. Despite this lack of detailed mechanistic understanding, here we show how a relatively simple model enables accurate and predictable tuning of synthetic gene expression system in yeast using several predic ...[more]

PMID: 24968317

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Project description:BackgroundSaccharomyces cerevisiae is the first eukaryotic organism for which a multi-compartment genome-scale metabolic model was constructed. Since then a sequence of improved metabolic reconstructions for yeast has been introduced. These metabolic models have been extensively used to elucidate the organizational principles of yeast metabolism and drive yeast strain engineering strategies for targeted overproductions. They have also served as a starting point and a benchmark for the reconstruction of genome-scale metabolic models for other eukaryotic organisms. In spite of the successive improvements in the details of the described metabolic processes, even the recent yeast model (i.e., iMM904) remains significantly less predictive than the latest E. coli model (i.e., iAF1260). This is manifested by its significantly lower specificity in predicting the outcome of grow/no grow experiments in comparison to the E. coli model.ResultsIn this paper we make use of the automated GrowMatch procedure for restoring consistency with single gene deletion experiments in yeast and extend the procedure to make use of synthetic lethality data using the genome-scale model iMM904 as a basis. We identified and vetted using literature sources 120 distinct model modifications including various regulatory constraints for minimal and YP media. The incorporation of the suggested modifications led to a substantial increase in the fraction of correctly predicted lethal knockouts (i.e., specificity) from 38.84% (87 out of 224) to 53.57% (120 out of 224) for the minimal medium and from 24.73% (45 out of 182) to 40.11% (73 out of 182) for the YP medium. Synthetic lethality predictions improved from 12.03% (16 out of 133) to 23.31% (31 out of 133) for the minimal medium and from 6.96% (8 out of 115) to 13.04% (15 out of 115) for the YP medium.ConclusionsOverall, this study provides a roadmap for the computationally driven correction of multi-compartment genome-scale metabolic models and demonstrates the value of synthetic lethals as curation agents.

Project description:BackgroundGene expression is a central process in all living organisms. Central questions in the field are related to the way the expression levels of genes are encoded in the transcripts and affect their evolution, and the potential to predict expression levels solely by transcript features. In this study we analyze S. cerevisiae, a model organism with the most abundant relevant cellular and genomic measurements, to evaluate the accuracy in which expression levels can be predicted by different parts of the transcript. To this end, we perform various types of regression analyses based on a total of 5323 features of the transcript. The main advantage of the proposed predictors over previous ones is related to the accurate and comprehensive definitions of the relevant transcript features, which are based on biophysical knowledge of the gene transcription and translation processes, their modeling and evolution.ResultsCross validation analyses of our predictors demonstrate that they achieve a correlation of 0.68/0.68/0.70/0.61/0.81 with mRNA levels, ribosomal density, protein levels, proteins per mRNA molecule (PPR), and ribosomal load (RL) respectively (all p-values <10(-140)). When we consider predictors that are based exclusively on the features related to different parts of the transcript (5'UTR, ORF, 3'UTR), the correlations with protein levels were 0.27/0.71/0.25 (all p-values <10(-5)), suggesting that the information in the UTRs is redundant, and features of the ORF alone yield similar predictions to the ones obtained based on the entire transcript.ConclusionsThe reported results demonstrate that in the analyzed model organism the expression levels of a gene are encoded in the transcript. Specifically, the prediction of a large fraction of the variance of the different gene expression steps based on transcript features alone is feasible in S. cerevisiae. We report dozens of novel transcript features related to expression levels predictions, demonstrating how such analyses can aid in understanding the gene expression process and its evolution, and how such predictors can be designed for other organisms in the future.

Dataset Information

Accurate, model-based tuning of synthetic gene expression using introns in S. cerevisiae.

Publications

Accurate, model-based tuning of synthetic gene expression using introns in S. cerevisiae.

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