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

Abstract

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.

Markov State Models to Study the Functional Dynamics of Proteins in the Wake of Machine Learning

“…AIP Publishing, 2020. Panels (C)–(E) are adapted with permission from ref ( 90 ). National Academy of Sciences, 2021.…”

“…Recently, qMSMs have been successfully applied to elucidate the dynamics of a large functional conformational change of the bacterial RNA Polymerase (RNAP) transcription complex: i.e., the opening of the RNAP clamp. 90 Bacterial RNAP has a shape that resembles a crab claw with two pincers: clamp and β-lobe (see yellow and magenta regions, respectively in Figure 5 C). The opening and closing of the clamp are crucial for the initiation of bacterial gene transcription, and inhibition of the RNAP clamp opening provides a promising target for the development of antibiotics (e.g., Myxopyronin).…”

Modeling DNA Opening in the Eukaryotic Transcription Initiation Complexes via Coarse-Grained Models

“…Once comprehensively sampled by CG-MD, one can back-map the sampled CG structure models into AA models, followed by AA-MD simulations ( Shimizu and Takada, 2018 ). A recent study employed such a protocol to gain comprehensive and high-resolution energy landscape in a bacterial RNA polymerase ( Unarta et al, 2021 ).…”

RPnet: A Reverse Projection Based Neural Network for Coarse-graining Metastable Conformational States for Protein Dynamics

“…For this system, the PCCA+ lumping is obtained from the crisp assignment of the PCCA+ fuzzy lumping 38 via the PyEMMA 33 software. When performing the kinetic lumping, we have adopted the same microstate assignments as Ref 58 but chose a slightly different lagtime of 90ns rather than the lag time of 60ns adopted in Ref 58 . The hierarchical clustering with Ward linkage is done based on the distance matrix , where t ij ′ is the element of the symmetrized TCM.…”

“…5a, the four macrostates of this system correspond to the open state (State S1, yellow dots), the closed state (State S4, green dots), as well as two partially closed states differing by the switch 2 region; partially closed with α -helix switch 2 (State S2, blue dots) and ߨ -helix switch 2 (State S3, red dots). The switch 2 region is a helical structure under the clamp domain which is crucial for the clamp movement 58 . In the PCCA+ based four-state model reported before, the transitions between S1/S2 or S3/S4 only takes 10~100 μ s, while the transitions between the two partially closed state S2/S3 would take 2 ms, indicating a huge gap between S2 and S3.…”