To initiate transcription, the holoenzyme (RNA polymerase [RNAP] in complex with σ factor) loads the promoter DNA via the flexible loading gate created by the clamp and β-lobe, yet their roles in DNA loading have not been characterized. We used a quasi-Markov State Model (qMSM) built from extensive molecular dynamics simulations to elucidate the dynamics of Thermus aquaticus holoenzyme's gate opening. We showed that during gate opening, β-lobe oscillates four orders of magnitude faster than the clamp, whose opening depends on the Switch 2's structure. Myxopyronin, an antibiotic that binds to Switch 2, was shown to undergo a conformational selection mechanism to inhibit clamp opening. Importantly, we reveal a critical but undiscovered role of β-lobe, whose opening is sufficient for DNA loading even when the clamp is partially closed. These findings open the opportunity for the development of antibiotics targeting β-lobe of RNAP. Finally, we have shown that our qMSMs, which encode non-Markovian dynamics based on the generalized master equation formalism, hold great potential to be widely applied to study biomolecular dynamics.
Keywords: Markov State Model; bacterial RNA polymerase; transcription initiation.