Recorded: 18/3/2026

Prof. Cesar Ramirez-Sarmiento (Pontificia Universidad Católica de Chile, Santiago, Chile)

Abstract:
RfaH, a two domain protein found in bacteria, is a transcription factor of the universally conserved NusG/Spt5 family and a quintessential example of a fold-switching protein. Its C-terminal switches from an α-helical hairpin bound to the N-terminal domain, into an active NusG-like state by C-terminal domain dissociation and refolding into a β-barrel. However, the emergence of this protein during evolution has been seldom explored. Here, we will show how large-scale protein structure prediction of RfaH homologs using AlphaFold2 enables to identify putative extant RfaH proteins that are constitutively folded in the active state and are functional. Then, using ancestral sequence reconstruction, we will showcase how RfaH evolved from early ancestors that already had fold-switching behaviors, as ascertained by protein structure predictions, biophysical and functional assays. These results illustrate how RfaH might have evolved from monomorphic ancestors into metamorphic ones to enable its action over operons far from the production site of RfaH in bacterial genomes