NusG is the only transcription factor present in every living cell. NusG associates with RNAP across most genes and interacts with other proteins to promote rRNA synthesis, coupled translation, or Rho-dependent termination. In Bacteria, diverse NusG paralogs are required for expression of long operons that encode biosynthesis of antibiotics, capsules, cell walls, and secretion systems. Among these proteins, E. coli RfaH is the best studied.
Unlike NusG, RfaH is recruited to RNAP only at specific ops DNA sequences, which halt RNAP and directly bind to RfaH. In free autoinhibited RfaH, the α-helical C-terminal domain (CTD) masks the RNAP-binding site on the N-terminal domain (NTD). Binding to the transcription complex activates RfaH: the domains dissociate, and the CTD refolds into a β-barrel. This dramatic transformation defies the one-fold—one-structure paradigm and is required for RfaH function. The α-CTD prevents off-target recruitment of RfaH to RNAP, which interferes with the essential function of NusG. The β-CTD recruits the ribosome, bypassing the need for Shine-Dalgarno element, a major component of gene activation by RfaH. Remarkably, RfaH transformation is fully reversible.
Protein metamorphosis is involved in diverse processes, from circadian clocks to neurodegenerative disorders, but metamorphic proteins are challenging to identify and study. We established RfaH as a paradigm to study metamorphosis. With Cesar Ramirez-Sarmiento, Stefan Knauer, and Markus Wahl, we are using NMR, cryo-EM, molecular dynamics simulations, mass spectrometry, and biochemical analyses to elucidate the details of RfaH transformation. Our results will guide discovery of natural transformer proteins and design of synthetic variants for biotechnological applications.