Protein-protein interaction specificity is often encoded at the primary sequence level. However, the contributions of individual residues to specificity are usually poorly understood and often obscured by mutational robustness, sequence degeneracy, and epistasis. Using bacterial toxin-antitoxin systems as a model, we screened a combinatorially complete library of antitoxin variants at three key positions against two toxins. This library enabled us to measure the effect of individual substitutions on specificity in hundreds of genetic backgrounds. These distributions allow inferences about the general nature of interface residues in promoting specificity. We find that positive and negative contributions to specificity are neither inherently coupled nor mutually exclusive. Further, a wild-type antitoxin appears optimized for specificity as no substitutions improve discrimination between cognate and non-cognate partners. By comparing crystal structures of paralogous complexes, we provide a rationale for our observations. Collectively, this work provides a generalizable approach to understanding the logic of molecular recognition.

The rewiring of gene regulatory networks can generate phenotypic novelty. It remains an open question, however, how the large number of connections needed to form a novel network arise over evolutionary time. Here, we address this question using the network controlled by the fungal transcription regulator Ndt80. This conserved protein has undergone a dramatic switch in function—from an ancestral role regulating sporulation to a derived role regulating biofilm formation. This switch in function corresponded to a large-scale rewiring of the genes regulated by Ndt80. However, we demonstrate that the Ndt80-target gene connections were undergoing extensive rewiring prior to the switch in Ndt80’s regulatory function. We propose that extensive drift in the Ndt80 regulon allowed for the exploration of alternative network structures without a loss of ancestral function, thereby facilitating the formation of a network with a new function.DOI: http://dx.doi.org/10.7554/eLife.23250.001

We examine how a complex transcription network composed of seven 'master' regulators and hundreds of target genes evolved over a span of approximately 70 million years. The network controls biofilm formation in several Candida species, a group of fungi that are present in humans both as constituents of the microbiota and as opportunistic pathogens. Using a variety of approaches, we observed two major types of changes that have occurred in the biofilm network since the four extant species we examined last shared a common ancestor. Master regulator 'substitutions' occurred over relatively long evolutionary times, resulting in different species having overlapping, but different sets of master regulators of biofilm formation. Second, massive changes in the connections between the master regulators and their target genes occurred over much shorter timescales. We believe this analysis is the first detailed, empirical description of how a complex transcription network has evolved.

The rewiring of gene regulatory networks over evolutionary timescales produces changes in the patterns of gene expression and is a major source of diversity among species. Yet the molecular mechanisms underlying evolutionary rewiring are only beginning to be understood. Here, we discuss recent analyses in ascomycete yeasts that have revealed several general principles of network rewiring. Specifically, we discuss how transcription networks can maintain a functional output despite changes in mechanism, how specific types of constraints alter available evolutionary trajectories, and how regulatory rewiring can ultimately lead to phenotypic novelty. We also argue that the structure and "logic" of extant gene regulatory networks can largely be accounted for by constraints that shape their evolutionary trajectories.

Toxin-antitoxin (TA) systems are widespread genetic modules found in almost all bacteria that can regulate their growth and may play prominent roles in phage defense. Escherichia coli encodes 11 TA systems in which the toxin is a known or predicted endoribonuclease. The targets and cleavage specificities of these endoribonucleases have remained largely uncharacterized, precluding an understanding of how each impacts cell growth and an assessment of whether they have distinct or overlapping targets.