Adaptation is less accessible through mutations in promoters than in coding sequences when large effect sizes are needed

Mutations within promoters and coding sequences are both involved in adaptation, but their relative contributions remain to be compared directly. Using the fungal enzyme cytosine deaminase, we examine if adaptation to 5-fluorocytosine (5-FC), which requires reduced cytosine deamination, can be reached by single promoter mutations. We generated all single-nucleotide substitutions and indels in the yeast FCY1 promoter and assayed the resulting mutants in presence of 5-FC. This revealed that no promoter mutation is sufficient for adaptation to occur. In contrast, 29% of single-nucleotide substitutions in the FCY1 gene have previously been reported as conferring adaptation to 5-FC. We hypothesized that this discrepancy could arise if FCY1 expression was robust to promoter mutations or if fitness was insensitive to expression level changes. Combining large-scale expression measurements with the experimental characterization of the corresponding expression-fitness function, we found that, while 24% of promoter mutations significantly affect expression, the shape of this function prevents them from being adaptive. Because this relationship is step-like, the curve is flat around wild-type level and adaptation can only emerge from a severe reduction of expression. These results highlight how adaptive phenotypes may be more mutationally accessible in coding sequences than promoters, especially when large effects are beneficial.