A general strategy to construct small molecule biosensors in eukaryotes
- Harvard Medical School, United States
- University of Washington, United States
- Colorado State University, United States
- Howard Hughes Medical Institute, University of Washington, United States
Abstract
Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.
Article and author information
Author details
Reviewing Editor
- Jeffery W Kelly, Scripps Research Institute, United States
Version history
- Received: August 6, 2015
- Accepted: December 17, 2015
- Accepted Manuscript published: December 29, 2015 (version 1)
- Accepted Manuscript updated: December 30, 2015 (version 2)
- Version of Record published: January 26, 2016 (version 3)
Copyright
© 2015, Feng et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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A general strategy to construct small molecule biosensors in eukaryotes
eLife 4:e10606.
https://doi.org/10.7554/eLife.10606
Further reading
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Previously, Tuller et al. found that the first 30–50 codons of the genes of yeast and other eukaryotes are slightly enriched for rare codons. They argued that this slowed translation, and was adaptive because it queued ribosomes to prevent collisions. Today, the translational speeds of different codons are known, and indeed rare codons are translated slowly. We re-examined this 5’ slow translation ‘ramp.’ We confirm that 5’ regions are slightly enriched for rare codons; in addition, they are depleted for downstream Start codons (which are fast), with both effects contributing to slow 5’ translation. However, we also find that the 5’ (and 3’) ends of yeast genes are poorly conserved in evolution, suggesting that they are unstable and turnover relatively rapidly. When a new 5’ end forms de novo, it is likely to include codons that would otherwise be rare. Because evolution has had a relatively short time to select against these codons, 5’ ends are typically slightly enriched for rare, slow codons. Opposite to the expectation of Tuller et al., we show by direct experiment that genes with slowly translated codons at the 5’ end are expressed relatively poorly, and that substituting faster synonymous codons improves expression. Direct experiment shows that slow codons do not prevent downstream ribosome collisions. Further informatic studies suggest that for natural genes, slow 5’ ends are correlated with poor gene expression, opposite to the expectation of Tuller et al. Thus, we conclude that slow 5’ translation is a ‘spandrel’--a non-adaptive consequence of something else, in this case, the turnover of 5’ ends in evolution, and it does not improve translation.
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