Rfah: Folding Switches in Transcription and Bacterial Physiology

Proteins that undergo structural rearrangements play key roles in cellular function, and one such example is RfaH, a bacterial transcription factor with the unusual ability to switch between distinct folds. This transformation allows it to dynamically modulate gene expression, influencing various physiological processes in bacteria. Understanding RfaH provides insight into broader mechanisms of transcriptional regulation and protein conformational flexibility.

Structural Arrangement

RfaH adopts two distinct conformations, each serving a unique function. In its inactive state, the C-terminal domain (CTD) assumes an α-helical fold, resembling the NusG family of transcription factors. This autoinhibited conformation prevents premature interaction with the transcription machinery. The α-helical CTD is tightly packed against the N-terminal domain (NTD), stabilizing the protein in an inactive state.

Upon recognizing a conserved operon-specific sequence known as an ops element, RfaH undergoes a dramatic structural rearrangement. The CTD refolds from an α-helical structure into a β-barrel configuration, a rare example of reversible fold-switching in proteins. This transition frees the NTD to bind RNA polymerase and exert its regulatory effects. The shift is not a localized adjustment but a complete reorganization of secondary and tertiary structure, demonstrating extreme protein plasticity.

The stability of these conformations is governed by distinct intramolecular interactions. In the α-helical state, hydrophobic contacts between the CTD and NTD maintain autoinhibition, while in the β-barrel state, new hydrogen bonds and β-strand interactions stabilize the alternative fold. Unlike many transcription factors that rely on allosteric modulation or post-translational modifications, RfaH regulates activity through an intrinsic fold-switching mechanism, making it a unique model for studying protein dynamics.

Reversible Fold-Switching Mechanism

RfaH’s ability to switch folds allows it to alternate between structurally distinct states based on molecular cues. This transformation is triggered by the recognition of an ops element in the DNA, recruiting RfaH to the transcription machinery. Upon binding RNA polymerase, the NTD undergoes a conformational shift that destabilizes the α-helical CTD, initiating its refolding into a β-barrel.

The transition is governed by a balance of intramolecular forces. In the autoinhibited state, hydrophobic interactions stabilize the compact association between the CTD and NTD. Upon ops element recognition, these contacts are disrupted, exposing residues that favor β-strand formation. Newly formed hydrogen bonds and β-sheet interactions reinforce the β-barrel state. Unlike conventional allosteric regulation, where ligand binding induces subtle conformational shifts, RfaH’s transformation involves a complete domain refolding. This extreme plasticity allows the protein to function as a binary molecular switch.

Reverting to the autoinhibited conformation requires assistance from cellular chaperones or specific molecular conditions. Recent studies suggest the β-barrel state may be inherently metastable, predisposing it to refold into its original α-helical configuration under the right conditions. This reversibility ensures RfaH remains responsive to environmental and cellular cues, allowing reuse without degradation or resynthesis.

Role In Transcription Control

RfaH selectively enhances the expression of operons involved in virulence, stress response, and extracellular factor production. Unlike general elongation factors, RfaH activates only a subset of genes containing an ops element. This specificity prevents unnecessary resource expenditure by the bacterial cell.

Recruitment of RfaH by RNA polymerase at ops-containing promoters stabilizes the transcription elongation complex, reducing premature termination and increasing RNA synthesis efficiency. This function is crucial for long, complex operons prone to transcriptional pausing.

RfaH stabilizes RNA polymerase by interacting with its β’ clamp domain, reinforcing its DNA association. This not only enhances transcription processivity but also shields the elongation complex from termination factors. The effect is most pronounced in genes encoding large, polycistronic transcripts, which require sustained elongation. In pathogens such as Escherichia coli and Yersinia pestis, RfaH facilitates the expression of virulence-associated operons, enabling bacteria to produce adhesins, toxins, and secretion systems essential for host colonization and immune evasion.

Beyond elongation, RfaH influences post-transcriptional events by modulating RNA stability and translation efficiency. The genes it regulates often encode functionally linked proteins, necessitating coordinated expression. By preventing premature termination, RfaH ensures full-length transcripts are available for translation, reducing the likelihood of truncated proteins. Additionally, the β-barrel form of the CTD resembles a translation initiation factor, potentially influencing ribosome recruitment. This dual role in transcription and translation underscores its broader impact on bacterial adaptability and survival.

Links To Bacterial Physiology

RfaH’s regulatory influence extends beyond transcription, shaping bacterial physiology to enhance survival, adaptation, and resource allocation. By promoting the expression of large, energetically demanding operons, RfaH helps bacteria optimize responses to environmental challenges. In nutrient-limited conditions, its activity ensures resources are directed toward functionally linked protein synthesis, improving metabolic efficiency.

RfaH also plays a key role in biofilm formation. Studies on Escherichia coli and Pseudomonas aeruginosa show that RfaH-dependent operons contribute to extracellular polysaccharide production, crucial for biofilm stability. By preventing transcription termination, RfaH facilitates the synthesis of these structural components, reinforcing bacterial communities against environmental stressors such as desiccation and antibiotic exposure. This regulation enhances bacterial persistence, particularly in clinical settings where biofilms complicate treatment strategies.

Analytical Methods

Studying RfaH’s structural and functional properties requires biochemical, biophysical, and genetic approaches. Because its reversible fold-switching mechanism is rare, specialized techniques capture both conformations and analyze their transitions in real time.

Structural studies rely on X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy to resolve the α-helical and β-barrel states. X-ray crystallography provides high-resolution snapshots of each conformation, revealing stabilizing interactions. NMR spectroscopy captures dynamic aspects of fold switching, offering insight into structural flexibility and transient intermediates. These methods confirm that RfaH undergoes a complete secondary structure reorganization rather than a localized shift.

Single-molecule fluorescence resonance energy transfer (smFRET) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) track the kinetics of RfaH’s fold-switching process. smFRET monitors conformational changes in real time, while HDX-MS provides information on solvent accessibility and hydrogen bonding patterns, shedding light on structural stability. These techniques reveal that RfaH’s switch is a highly coordinated process. Mutagenesis studies further identify residues critical for maintaining each fold, offering potential targets for modulating RfaH activity in bacterial systems.