A major role for noncoding regulatory mutations in the evolution of enzyme activity

Abstract

The quantitative evolution of protein activity is a common phenomenon, yet we know little about any general mechanistic tendencies that underlie it. For example, an increase (or decrease) in enzyme activity may evolve from changes in protein sequence that alter specific activity, or from changes in gene expression that alter the amount of protein produced. The latter in turn could arise via mutations that affect gene transcription, posttranscriptional processes, or copy number. Here, to determine the types of genetic changes underlying the quantitative evolution of protein activity, we dissected the basis of ecologically relevant differences in Alcohol dehydrogenase (Adh) enzyme activity between and within several Drosophila species. By using recombinant Adh transgenes to map the functional divergence of ADH enzyme activity in vivo, we find that amino acid substitutions explain only a minority (0 to 25%) of between- and within-species differences in enzyme activity. Instead, noncoding substitutions that occur across many parts of the gene (enhancer, promoter, and 5′ and 3′ untranslated regions) account for the majority of activity differences. Surprisingly, one substitution in a transcriptional Initiator element has occurred in parallel in two species, indicating that core promoters can be an important natural source of the tuning of gene activity. Furthermore, we show that both regulatory and coding substitutions contribute to fitness (resistance to ethanol toxicity). Although qualitative changes in protein specificity necessarily derive from coding mutations, these results suggest that regulatory mutations may be the primary source of quantitative changes in protein activity, a possibility overlooked in most analyses of protein evolution.

Fitness effects but no temperature-mediated balancing selection at the polymorphic Adh gene of Drosophila melanogaster

“…Linked nucleotide polymorphisms in the Fast allele therefore increase ethanol tolerance only in adults, in contrast to amino acid polymorphism T192, which increases ethanol tolerance only in larvae. The transgenic Fast allele we used has been shown to increase expression compared to the Slow allele, and the alcohol dehydrogenase activity in lysate from these flies is similar to that of natural Fast and Slow strains, respectively (68). Both coding and regulatory aspects of the Fast allele therefore contribute to increased ethanol resistance, at different life stages.…”

“…We tested this possibility by engineering transgenic flies containing a naturally occurring Fast Adh allele, which corresponds precisely in its boundaries to that of our Slow and Slow+T192 alleles. The Fast allele construct comprises the entire coding sequence, all introns, and flanking regions that include all previously characterized cis -regulatory regions known to affect Adh expression, including the Δ1 indel polymorphism that cosegregates with T192 (49, 50, 68). We compared ethanol resistance of these transgenic Fast-allele flies at varying temperature to those carrying the Slow and Slow+T192 alleles described above.…”

The origin and diversification of a novel protein family in venomous snakes

“…We would like to draw attention to recent work from this laboratory which reported two pertinent observations from the analysis of the evolution of increased alcohol dehydrogenase (Adh) gene activity in certain Drosophila species. Specifically, it was found that the expression of tandem gene duplicates is often greater than twofold that of single genes (49) and that a tandem gene duplication produced the single largest quantitative effect on Adh protein activity of a variety of individual mutations among several Drosophila species (49). The relevant implication of these studies is that selection for greater gene expression is sufficient to favor the retention of a gene duplicate without any prior or subsequent innovative mutations.…”

Adaptive substitutions underlying cardiac glycoside insensitivity in insects exhibit epistasis in vivo

“…Another alternative is that protein dysfunction associated with CG-insensitivity substitutions is in some cases compensated by the up-regulation of ATPα1 expression (see for e.g. Loehlin et al, 2019), which may be an important driver of recurrent ATPα1 duplication and is observed across multiple CG-adapted insects (Yang et al, 2019).…”