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Structural fluctuations and mechanical stabilities of the metamorphic protein RfaH.

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The bacterial transcription factor RfaH undergoes a dramatic structural change in its C-terminal domain (CTD) upon binding RNA polymerase (RNAP). Interdomain contacts stabilize the inactive RfaH, while mechanical properties reveal distinct rigid and soft regions.

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Monte Carlofold switchingmechanical fingerprintingmechanical unfoldingprotein foldingtransformer protein

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Area of Science:

  • Molecular Biology
  • Structural Biology
  • Biophysics

Background:

  • RfaH is a bacterial transcription factor with dual roles in transcription regulation and translation enhancement.
  • Its function relies on a significant fold switch in the C-terminal domain (CTD), transitioning from an alpha-helical to a beta-sheet state.
  • This transformation is triggered by binding to RNA polymerase (RNAP), but the precise mechanism remains unclear.

Purpose of the Study:

  • To investigate the structural fluctuations and mechanical stability of RfaH and its isolated CTD.
  • To elucidate the role of interdomain contacts in maintaining the CTD's structure.
  • To understand how mechanical properties might facilitate the RfaH fold switch upon RNAP interaction.

Main Methods:

  • All-atom Monte Carlo simulations were employed to study RfaH and its CTD.
  • Pulling simulations were used to measure mechanical stability by quantifying deformation resistance.

Main Results:

  • Simulations confirmed that interdomain contacts are essential for the stability of the all-alpha CTD in free RfaH.
  • A mechanical stability profile revealed that RfaH can be divided into distinct rigid and soft regions.
  • The boundary between these regions closely aligns with the interface between RfaH's two domains.

Conclusions:

  • Interdomain contacts play a critical role in stabilizing the inactive, all-alpha conformation of the RfaH CTD.
  • The identified mechanical properties, particularly the rigid-soft domain boundary, may provide a mechanism for triggering the CTD fold switch upon RNAP binding.
  • This study offers insights into the molecular basis of RfaH's dual function and its regulation by RNAP.