Rescue of Escherichia coli auxotrophy by de novo small proteins
- Uppsala University, Sweden
- European Molecular Biology Laboratory, Germany
Abstract
Increasing numbers of small proteins with diverse physiological roles are being identified and characterized in both prokaryotic and eukaryotic systems, but the origins and evolution of these proteins remain unclear. Recent genomic sequence analyses in several organisms suggest that new functions encoded by small open reading frames (sORFs) may emerge de novo from noncoding sequences. However, experimental data demonstrating if and how randomly generated sORFs can confer beneficial effects to cells are limited. Here we show that by up-regulating hisB expression, de novo small proteins (≤ 50 amino acids in length) selected from random sequence libraries can rescue Escherichia coli cells that lack the conditionally essential SerB enzyme. The recovered small proteins are hydrophobic and confer their rescue effect by binding to the 5' end regulatory region of the his operon mRNA, suggesting that protein binding promotes structural rearrangements of the RNA that allow increased hisB expression. This study adds RNA regulatory elements as another interacting partner for de novo proteins isolated from random sequence libraries, and provides further experimental evidence that small proteins with selective benefits can originate from the expression of nonfunctional sequences.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting file. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (Perez-Riverol et al. 2019) with the dataset identifier PXD014049.
Article and author information
Author details
Funding
Knut och Alice Wallenbergs Stiftelse (2015.0069)
- Dan I Andersson
Vetenskapsrådet (2017-01527)
- Dan I Andersson
Vetenskapsrådet (2019-00666)
- Michael Knopp
Vetenskapsrådet (2020‐04395)
- Per Jemth
Knut och Alice Wallenbergs Stiftelse (2017.0071)
- Mårten Larsson
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Joseph T Wade, New York State Department of Health, United States
Version history
- Received: March 1, 2022
- Preprint posted: April 15, 2022 (view preprint)
- Accepted: March 14, 2023
- Accepted Manuscript published: March 15, 2023 (version 1)
- Version of Record published: March 31, 2023 (version 2)
Copyright
© 2023, Babina 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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Rescue of Escherichia coli auxotrophy by de novo small proteins
eLife 12:e78299.
https://doi.org/10.7554/eLife.78299
Further reading
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- Biochemistry and Chemical Biology
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The emergence of new protein functions is crucial for the evolution of organisms. This process has been extensively researched for soluble enzymes, but it is largely unexplored for membrane transporters, even though the ability to acquire new nutrients from a changing environment requires evolvability of transport functions. Here, we demonstrate the importance of environmental pressure in obtaining a new activity or altering a promiscuous activity in members of the amino acid-polyamine-organocation (APC)-type yeast amino acid transporters family. We identify APC members that have broader substrate spectra than previously described. Using in vivo experimental evolution, we evolve two of these transporter genes, AGP1 and PUT4, toward new substrate specificities. Single mutations on these transporters are found to be sufficient for expanding the substrate range of the proteins, while retaining the capacity to transport all original substrates. Nonetheless, each adaptive mutation comes with a distinct effect on the fitness for each of the original substrates, illustrating a trade-off between the ancestral and evolved functions. Collectively, our findings reveal how substrate-adaptive mutations in membrane transporters contribute to fitness and provide insights into how organisms can use transporter evolution to explore new ecological niches.
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