The evolution of function in the DNA binding domain of the CRP/FNR family

Transcriptional regulation, facilitated by transcription factors (TFs), contributes to bacterial response to environmental and cellular perturbations. How have TFs diversified and gained their functions? We use the CRP/FNR family to study this question across [~]6000 prokaryotic genomes. Characterized by homology to TFs CRP (cyclic AMP Receptor Protein) and FNR (Regulator of Fumarate and Nitrate Reductase) in Escherichia coli, the two functions of this family - sequence-specific DNA binding and transcriptional activation by direct contacts to RNA polymerase - are conferred by the helix-turn-helix containing DNA binding domain. We constructed a rooted phylogeny of this domain and performed residue conservation analysis on extant and in-silico reconstructed ancestral sequences of CRP/FNR family members. Residues that confer DNA binding specificity likely emerged in the overall ancestor of the sequence family. This ancestor does not fall into classes containing either E. coli CRP (Eco-CRP) or E. coli FNR (Eco-FNR) clades, but instead a large and diverse class that we call "CRP-like". Residues key for base-specific DNA binding remain largely conserved in the family. Residues (together called AR1) which make direct contacts with CTD of RNA Polymerase are conserved only in the Eco-CRP class and restricted to Gammaproteobacteria. The corresponding 287 determinant interface on CTD is again fully conserved only in Gammaproteobacteria, which contain Eco-CRP members, following step-wise evolution initiated at the Proteobacterial common ancestor. These suggest suggesting co-emergence of the interface residues in Eco-CRP. Our analysis hence shows that Class I transcriptional activation through AR1 on CRP and the determinant of CTD, as described in E. coli CRP, is phylogenetically restricted, while base-specific DNA binding has been present in the CRP/FNR family throughout its evolutionary history.