Role of bacterial RNA polymerase gate opening dynamics in DNA loading and antibiotics inhibition elucidated by quasi-Markov State Model

To initiate transcription, the holoenzyme (RNA polymerase [RNAP] in complex with sigma factor) loads the promoter DNA via the flexible loading gate created by the clamp and beta-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, beta-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 beta-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 beta-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.

Automated summary

The study elucidated the dynamics of Thermus aquaticus holoenzyme's gate opening using a quasi-Markov State Model, revealing the differential roles of beta-lobe and clamp in DNA loading and the mechanism by which Myxopyronin inhibits clamp opening. Additionally, a critical role of beta-lobe in DNA loading was uncovered, presenting an opportunity for the development of antibiotics targeting this region of RNAP. The study also demonstrated the potential of quasi-Markov State Models in studying biomolecular dynamics based on generalized master equation formalism.