Reversible fold-switching controls the functional cycle of the antitermination factor RfaH.
ABSTRACT: RfaH, member of the NusG/Spt5 family, activates virulence genes in Gram-negative pathogens. RfaH exists in two states, with its C-terminal domain (CTD) folded either as α-helical hairpin or β-barrel. In free RfaH, the α-helical CTD interacts with, and masks the RNA polymerase binding site on, the N-terminal domain, autoinhibiting RfaH and restricting its recruitment to opsDNA sequences. Upon activation, the domains separate and the CTD refolds into the β-barrel, which recruits a ribosome, activating translation. Using NMR spectroscopy, we show that only a complete ops-paused transcription elongation complex activates RfaH, probably via a transient encounter complex, allowing the refolded CTD to bind ribosomal protein S10. We also demonstrate that upon release from the elongation complex, the CTD transforms back into the autoinhibitory α-state, resetting the cycle. Transformation-coupled autoinhibition allows RfaH to achieve high specificity and potent activation of gene expression.
Project description: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 ?-helical hairpin, which binds to the N-terminal domain (NTD) in the free protein, to a ?-barrel, which interacts with the ribosomal protein S10 following RfaH recruitment to its target operons. The CTD forms a ?-barrel when expressed alone or proteolytically separated from the NTD, indicating that the ?-helical state is trapped by the NTD, perhaps co-translationally. Alternatively, the interdomain contacts may be sufficient to drive the formation of the ?-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.
Project description:NusG homologs regulate transcription and coupled processes in all living organisms. The Escherichia coli (E. coli) two-domain paralogs NusG and RfaH have conformationally identical N-terminal domains (NTDs) but dramatically different carboxy-terminal domains (CTDs), a ? barrel in NusG and an ? hairpin in RfaH. Both NTDs interact with elongating RNA polymerase (RNAP) to reduce pausing. In NusG, NTD and CTD are completely independent, and NusG-CTD interacts with termination factor Rho or ribosomal protein S10. In contrast, RfaH-CTD makes extensive contacts with RfaH-NTD to mask an RNAP-binding site therein. Upon RfaH interaction with its DNA target, the operon polarity suppressor (ops) DNA, RfaH-CTD is released, allowing RfaH-NTD to bind to RNAP. Here, we show that the released RfaH-CTD completely refolds from an all-? to an all-? conformation identical to that of NusG-CTD. As a consequence, RfaH-CTD binding to S10 is enabled and translation of RfaH-controlled operons is strongly potentiated. PAPERFLICK:
Project description:Proteins such as the transcription factor RfaH can change biological function by switching between distinct three-dimensional folds. RfaH regulates transcription if the C-terminal domain folds into a double helix bundle and promotes translation when this domain assumes a ?-barrel form. This fold-switch has been also observed for the isolated C-terminal domain, dubbed by us as RfaH-C-terminal domain (RfaH-CTD), and is studied here with a variant of the replica-exchange-with-tunneling approach recently introduced by us. We use the enhanced sampling properties of this technique to map the free-energy landscape of RfaH-CTD and to propose a mechanism for the conversion process.
Project description:RfaH is a virulence factor from Escherichia coli whose C-terminal domain (CTD) undergoes a dramatic ?-to-? conformational transformation. The CTD in its ?-helical fold is stabilized by interactions with the N-terminal domain (NTD), masking an RNA polymerase binding site until a specific recruitment site is encountered. Domain dissociation is triggered upon binding to DNA, allowing the NTD to interact with RNA polymerase to facilitate transcription while the CTD refolds into the ?-barrel conformation that interacts with the ribosome to activate translation. However, structural details of this transformation process in the context of the full protein remain to be elucidated. Here, we explore the mechanism of the ?-to-? conformational transition of RfaH in the full-length protein using a dual-basin structure-based model. Our simulations capture several features described experimentally, such as the requirement of disruption of interdomain contacts to trigger the ?-to-? transformation, confirms the roles of previously indicated residues E48 and R138, and suggests a new important role for F130, in the stability of the interdomain interaction. These native basins are connected through an intermediate state that builds up upon binding to the NTD and shares features from both folds, in agreement with previous in silico studies of the isolated CTD. We also examine the effect of RNA polymerase binding on the stabilization of the ? fold. Our study shows that native-biased models are appropriate for interrogating the detailed mechanisms of structural rearrangements during the dramatic transformation process of RfaH.
Project description:RfaH is a bacterial elongation factor that increases expression of distal genes in several long, horizontally acquired operons. RfaH is recruited to the transcription complex during RNA chain elongation through specific interactions with a DNA element called ops. Following recruitment, RfaH remains bound to RNA polymerase (RNAP) and acts as an antiterminator by reducing RNAP pausing and termination at some factor-independent and Rho-dependent signals. RfaH consists of two domains connected by a flexible linker. The N-terminal RfaH domain (RfaH(N)) recognizes the ops element, binds to the RNAP and reduces pausing and termination in vitro. Functional analysis of single substitutions in this domain reported here suggests that three separate RfaH(N) regions mediate these functions. We propose that a polar patch on one side of RfaH(N) interacts with the non-template DNA strand during recruitment, whereas a hydrophobic surface on the opposite side of RfaH(N) remains bound to the beta' subunit clamp helices domain throughout transcription of the entire operon. The third region is apparently dispensable for RfaH binding to the transcription complex but is required for the antitermination modification of RNAP.
Project description:Efficient transcription of long polycistronic operons in bacteria frequently relies on accessory proteins but their molecular mechanisms remain obscure. RfaH is a cellular elongation factor that acts as a polarity suppressor by increasing RNA polymerase (RNAP) processivity. In this work, we provide evidence that RfaH acts by reducing transcriptional pausing at certain positions rather than by accelerating RNAP at all sites. We show that 'fast' RNAP variants are characterized by pause-free RNA chain elongation and are resistant to RfaH action. Similarly, the wild-type RNAP is insensitive to RfaH in the absence of pauses. In contrast, those enzymes that may be prone to falling into a paused state are hypersensitive to RfaH. RfaH inhibits pyrophosphorolysis of the nascent RNA and reduces the apparent Michaelis-Menten constant for nucleotides, suggesting that it stabilizes the post-translocated, active RNAP state. Given that the RfaH-binding site is located 75 A away from the RNAP catalytic center, these results strongly indicate that RfaH acts allosterically. We argue that despite the apparent differences in the nucleic acid targets, the time of recruitment and the binding sites on RNAP, unrelated antiterminators (such as RfaH and lambdaQ) utilize common strategies during both recruitment and anti-pausing modification of the transcription complex.
Project description: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.
Project description:The only universally conserved family of transcription factors comprises housekeeping regulators and their specialized paralogs, represented by well-studied NusG and RfaH. Despite their ubiquity, little information is available on the evolutionary origins, functions, and gene targets of the NusG family members. We built a hidden Markov model profile of RfaH and identified its homologs in sequenced genomes. While NusG is widespread among bacterial phyla and coresides with genes encoding RNA polymerase and ribosome in all except extremely reduced genomes, RfaH is mostly limited to Proteobacteria and lacks common gene neighbors. RfaH activates only a few xenogeneic operons that are otherwise silenced by NusG and Rho. Phylogenetic reconstructions reveal extensive duplications and horizontal transfer of rfaH genes, including those borne by plasmids, and the molecular evolution pathway of RfaH, from "early" exclusion of the Rho terminator and tightened RNA polymerase binding to "late" interactions with the ops DNA element and autoinhibition, which together define the RfaH regulon. Remarkably, NusG is not only ubiquitous in Bacteria but also common in plants, where it likely modulates the transcription of plastid genes.IMPORTANCE In all domains of life, NusG-like proteins make contacts similar to those of RNA polymerase and promote pause-free transcription yet may play different roles, defined by their divergent interactions with nucleic acids and accessory proteins, in the same cell. This duality is illustrated by Escherichia coli NusG and RfaH, which silence and activate xenogenes, respectively. We combined sequence analysis and recent functional and structural insights to envision the evolutionary transformation of NusG, a core regulator that we show is present in all cells using bacterial RNA polymerase, into a virulence factor, RfaH. Our results suggest a stepwise conversion of a NusG duplicate copy into a sequence-specific regulator which excludes NusG from its targets but does not compromise the regulation of housekeeping genes. We find that gene duplication and lateral transfer give rise to a surprising diversity within the only ubiquitous family of transcription factors.
Project description:RfaH is required for virulence in several Gram-negative pathogens including Escherichia coli and Klebsiella pneumoniae. Through direct interactions with RNA polymerase (RNAP) and ribosome, RfaH activates the expression of capsule, cell wall and pilus biosynthesis operons by reducing transcription termination and activating translation. While E. coli RfaH has been extensively studied using structural and biochemical approaches, limited data are available for other RfaH homologs. Here we set out to identify small molecule inhibitors of E. coli and K. pneumoniae RfaHs. Results of biochemical and functional assays show that these proteins act similarly, with a notable difference between their interactions with the RNAP ? subunit gate loop. We focused on high-affinity RfaH interactions with the RNAP ?' subunit clamp helices as a shared target for inhibition. Among the top 10 leads identified by in silico docking using ZINC database, 3 ligands were able to inhibit E. coli RfaH recruitment in vitro. The most potent lead was active against both E. coli and K. pneumoniae RfaHs in vitro. Our results demonstrate the feasibility of identifying RfaH inhibitors using in silico docking and pave the way for rational design of antivirulence therapeutics against antibiotic-resistant pathogens.
Project description:The phase variation (reversible on-off switching) of the type 1 fimbrial adhesin of Escherichia coli involves a DNA inversion catalyzed by FimB (switching in either direction) or FimE (on-to-off switching). Here, we demonstrate that RfaH activates expression of a FimB-LacZ protein fusion while having a modest inhibitory effect on a comparable fimB-lacZ operon construct and on a FimE-LacZ protein fusion, indicating that RfaH selectively controls fimB expression at the posttranscriptional level. Further work demonstrates that loss of RfaH enables small RNA (sRNA) MicA inhibition of fimB expression even in the absence of exogenous inducing stress. This effect is explained by induction of ?(E), and hence MicA, in the absence of RfaH. Additional work confirms that the procaine-dependent induction of micA requires OmpR, as reported previously (A. Coornaert et al., Mol. Microbiol. 76:467-479, 2010, doi:10.1111/j.1365-2958.2010.07115.x), but also demonstrates that RfaH inhibition of fimB transcription is enhanced by procaine independently of OmpR. While the effect of procaine on fimB transcription is shown to be independent of RcsB, it was found to require SlyA, another known regulator of fimB transcription. These results demonstrate a complex role for RfaH as a regulator of fimB expression.