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Knowledge of the distribution of fitness effects (DFE) of mutations is critical to the understanding of protein evolution. Here, we describe methods for large-scale, systematic measurements of the DFE using growth competition and deep mutational scanning. We discuss techniques for producing comprehensive libraries of gene variants as well as provide necessary considerations for designing these experiments. Using these methods, we have constructed libraries containing over 18,000 variants, measured fitness effects of these mutations by deep mutational scanning, and verified the presence of fitness effects in individual variants. Our methods provide a high-throughput protocol for measuring biological fitness effects of mutations and the dependence of fitness effects on the environment.
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Fitness and functional landscapes of the E. coli RNase III gene rnc
Abstract How protein properties such as protein activity and protein essentiality affect the distribution of fitness effects (DFE) of mutations are important questions in protein evolution. Deep mutational scanning studies typically measure the effects of a comprehensive set of mutations on either protein activity or fitness. Our understanding of the underpinnings of the DFE would be enhanced by a comprehensive study of both for the same gene. Here, we compared the fitness effects and in vivo protein activity effects of ∼4,500 missense mutations in the E. coli rnc gene. This gene encodes RNase III, a global regulator enzyme that cleaves diverse RNA substrates including precursor ribosomal RNA and various mRNAs including its own 5’ untranslated region (5’UTR). We find that RNase III’s ability to cleave dsRNA is the most important determinant of the fitness effects of rnc mutations. The DFE of RNase III was bimodal, with mutations centered around neutral and deleterious effects, consistent with previously reported DFE’s of enzymes with a singular physiological role. Fitness was buffered to small effects on RNase III activity. The enzyme’s RNase III domain (RIIID), which contains the RNase III signature motif and all active site residues, was more sensitive to mutation than its dsRNA binding domain (dsRBD), which is responsible for recognition and binding to dsRNA. Differential effects on fitness and functional scores for mutations at highly conserved residues G97, G99, and F188 suggest that these positions may be important for RNase III cleavage specificity.
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- PAR ID:
- 10402609
- Editor(s):
- Echave, Julian
- Date Published:
- Journal Name:
- Molecular Biology and Evolution
- ISSN:
- 0737-4038
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1093/molbev/msad047
