Reversible fold-switching controls the functional cycle of the antitermination factor RfaH

Area of Science:

• Molecular Biology
• Microbiology
• Structural Biology

Background:

• RfaH (required forboldsymbolA function H) is a key regulator of virulence gene expression in Gram-negative pathogens.
• RfaH belongs to the NusG/Spt5 family and modulates transcription-translation coupling.
• The protein exists in two conformational states, regulated by its C-terminal domain (CTD).

Purpose of the Study:

• To elucidate the activation mechanism of RfaH by transcription complexes.
• To understand the structural transformations of the RfaH CTD during activation.
• To investigate the role of autoinhibition in RfaH function.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy was employed to study RfaH structure and interactions.
• Analysis of RfaH conformational changes in response to transcription elongation complexes.
• Biochemical assays to assess RfaH binding to ribosomal components.

Main Results:

• RfaH activation requires a complete ops-paused transcription elongation complex.
• The CTD refolds from an autoinhibitory α-helical state to a β-barrel conformation upon activation.
• The refolded CTD binds ribosomal protein S10, facilitating translation initiation.
• Released RfaH CTD reverts to the autoinhibitory α-state, resetting the regulatory cycle.

Conclusions:

• RfaH activation is a transformation-coupled process dependent on specific transcription intermediates.
• Autoinhibition mediated by the CTD ensures high specificity and potent activation of virulence gene expression.
• The RfaH mechanism provides a model for regulated transcription-translation coupling in bacteria.