coli main chromosome (ecDHFR) that is readily inhibited by TMP, DfrB1 is essentially insensitive to this antibiotic (DfrB1 K i ~ 0.15 mM Fig. 1B) ( 12, 13). Contrary to the monomeric FolA-type dihydrofolate reductase encoded in the E. The dihydrofolate reductase DfrB1 (formerly known as R67 DHFR) was first isolated as one of the factors causing trimethoprim (TMP) resistance in Enterobacteria, including Escherichia coli ( 11). We used a metabolic enzyme that produces a metabolite that is conditionally essential for growth ( Fig. 1A). We investigated the extent to which promoter activity affects the fitness landscape of a protein by mimicking evolutionary changes throughmutations affecting promoter activity using an inducible system. RESULTS The fitness landscape of a protein complex depends on its promoter activity level Here, we use deep mutational scanning (DMS) to examine how regulatory-by-coding epistasis could affect the evolutionary potential of a protein by performing a comprehensive measurement of the fitness effects of coding mutations at five levels of promoter activity in the vicinity of an expression level that is optimal for the wild-type (WT) enzyme. Thus, interactions between changes in gene expression and coding mutations could represent a vast underexplored constraint on protein evolution. ![]() For example, some disease-associated mutations have a higher penetrance when linked to a regulatory variant that increases their expression ( 10). Conversely, mutations that are deleterious at low expression levels could become neutral at higher expression levels or vice versa. ![]() ![]() For instance, the relative effects of coding mutations could be amplified at low expression level, as demonstrated in the study of a short amino acid segment of the heat shock protein Hsp90 ( 9). Unfortunately, both types of mutations are rarely studied simultaneously when mapping the effects of mutations on fitness, apart from a few cases ( 6– 9) revealing that epistasis between mutations affecting the expression of a gene and coding mutations (regulatory-by-coding) is likely frequent. Regulatory and coding changes underlie phenotypic variation within and between species ( 4, 5). We thereby demonstrate that promoter activity in interaction with protein properties can dictate which coding mutations are beneficial, neutral, or deleterious. Coding mutations that increase protein abundance are beneficial at low expression but could potentially incur a cost at high promoter activity. Mutations that are deleterious at low activity but masked at optimal activity are slightly destabilizing for individual subunits and binding interfaces. By measuring the fitness effects of all possible mutations on a protein complex at various levels of promoter activity, we show that promoter activity at the optimal level for the wild-type protein masks the effects of both deleterious and beneficial coding mutations. The extent and impact of the connection between these two dimensions are largely unknown because they have generally been studied independently. Menos and Velvet are married and one last action must be done.The evolution of protein-coding genes proceeds as mutations act on two main dimensions: regulation of transcription level and the coding sequence. FennFeatherDragon Fandoms: evoland, evoland 2ĪU to the Evoland 2 storyline.
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