Abstract
The initiation of bacterial transcription requires DNA loading via flexible loading gate of the RNA polymerase complex, consisting of the clamp and β-lobe domain. While the clamp is important in transcription initiation, the role of β-lobe remains unclear. Using quasi-Markov State Models (qMSMs) constructed from molecular dynamics simulations, we showed that the opening of β-lobe is orders of magnitude faster than that of the clamp, which depends on the structure of Switch 2. Strikingly, opening of the β-lobe is sufficient geometrically to accommodate DNA loading even when the clamp is partially closed. These two observations highlight β-lobe’s critical role allowing DNA loading during initiation. Thus, β-lobe is a promising target for antibiotics. qMSM also provides a promising tool investigating biomolecular dynamics.
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.