title: Interdomain contacts control folding of transcription factor RfaH.
abstract: Escherichia coli RfaH activates gene expression by tethering the elongating RNA polymerase to the ribosome. This bridging action requires a complete refolding of the RfaH C-terminal domain (CTD) from an α-helical hairpin, which binds to the N-terminal domain (NTD) in the free protein, to a β-barrel, which interacts with the ribosomal protein S10 following RfaH recruitment to its target operons. The CTD forms a β-barrel when expressed alone or proteolytically separated from the NTD, indicating that the α-helical state is trapped by the NTD, perhaps co-translationally. Alternatively, the interdomain contacts may be sufficient to drive the formation of the α-helical form. Here, we use functional and NMR analyses to show that the denatured RfaH refolds into the native state and that RfaH in which the order of the domains is reversed is fully functional in vitro and in vivo. Our results indicate that all information necessary to determine its fold is encoded within RfaH itself, whereas accessory factors or sequential folding of NTD and CTD during translation are dispensable. These findings suggest that universally conserved RfaH homologs may change folds to accommodate diverse interaction partners and that context-dependent protein refolding may be widespread in nature.
authors.affiliation: Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA, The Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA and Lehrstuhl Biopolymere und Forschungszentrum für Bio-Makromoleküle, Universität Bayreuth, Universitätsstraße 30, 95447 Bayreuth, Germany.
authors.forename: Sushil Kumar
authors.initials: SK
authors.lastname: Tomar
authors.forename: Stefan H
authors.initials: SH
authors.lastname: Knauer
authors.forename: Monali
authors.initials: M
authors.lastname: Nandymazumdar
authors.forename: Paul
authors.initials: P
authors.lastname: Rösch
authors.forename: Irina
authors.initials: I
authors.lastname: Artsimovitch
mesh_terms: D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D010445:Peptide Elongation Factors / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D058849:Protein Refolding; D017434:Protein Structure, Tertiary; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D010445:Peptide Elongation Factors; C106401:RfaH protein, E coli; D015534:Trans-Activators