Structural Basis for Converting a General Transcription Factor into an Operon-Specific Virulence Regulator

Abstract

RfaH, a paralog of the general transcription factor NusG, is recruited to elongating RNA polymerase at specific regulatory sites. The X-ray structure of Escherichia coli RfaH reported here reveals two domains. The N-terminal domain displays high similarity to that of NusG. In contrast, the alpha-helical coiled-coil C domain, while retaining sequence similarity, is strikingly different from the beta barrel of NusG. To our knowledge, such an all-beta to all-alpha transition of the entire domain is the most extreme example of protein fold evolution known to date. Both N domains possess a vast hydrophobic cavity that is buried by the C domain in RfaH but is exposed in NusG. We propose that this cavity constitutes the RNA polymerase-binding site, which becomes unmasked in RfaH only upon sequence-specific binding to the nontemplate DNA strand that triggers domain dissociation. Finally, we argue that RfaH binds to the beta' subunit coiled coil, the major target site for the initiation sigma factors.

The elongation factor RfaH and the initiation factor σ bind to the same site on the transcription elongation complex

“…Upon dissociation, the full-length RfaH switches into the inactive ''closed'' state, in which its two domains are tightly bound to each other (5), and cannot rebind the RNAP that has moved beyond the ops site. In vivo, other proteins may interact with the C-domain, precluding its reassociation with the N-domain and favoring stable binding of RfaH to the TEC.…”

“…DNA base pair and the first unpaired NT strand nucleotide in the transcription bubble: the TEC structure and a recent biochemical study (26) indicate the 9-bp RNA/DNA hybrid, with only one DNA base pair melted in the active site. The model of the RfaH N-domain was fitted to the tip of the CH as described previously (5). The TEC/ model was generated through superposition of the ␤Ј CH domain in the holoenzyme (3) with that in the TEC (25); the CH appears substantially displaced (by Ϸ7 Å) toward the main channel in the latter structure.…”

“…All plasmid constructs used in this work are listed in SI Table 1. RfaH variants, 70 , and altered RNAPs were purified as described previously (5). 38 was a gift of Jay Gralla (University of California, Los Angeles, CA).…”

“…Second, RfaH reduces pausing at all sites in vitro (7) yet increases pausing at the ops element located downstream from the identical ops site that mediates RfaH recruitment to the TEC (our unpublished observations), indicating that the N-domain retains the ability to interact with ops. Third, the RNAP variant missing the CH tip fails to respond to either full-length RfaH or the N-domain (5), arguing that the contacts to the CH are required regardless of the recruitment mechanism. Last, RfaH does not dissociate from the RNAP after its recruitment to ops (our unpublished observations).…”

“…In vitro, the ops element indeed mediates RfaH binding to the TEC but only if it is placed in the nontemplate (NT) DNA strand exposed on the surface of RNAP (7). RfaH recruitment is thought to occur in two steps: (i) sequence-specific binding of the N-terminal domain to DNA triggers displacement of the stably bound C-terminal domain to expose the RNAP binding site on the N-domain, and (ii) interactions of the N-domain with ␤Ј CH on one side and (nonspecific) interactions with the NT strand on the other allow for the stable retention of RfaH on the TEC throughout elongation (5).…”