Quasi Markov state models elucidates the role of bacterial RNA polymerase loading gate and trigger loop dynamics in antibiotics inhibition

The rise of antibiotic resistance urges the development of new antibiotics. Bacterial RNA Polymerase (RNAP) transcribes DNA to RNA and is an effective target for antibiotics. RNAP has a crab-claw-like shape with two pincers, clamp and β-lobe domains, forming a loading gate. During the initiation of transcription,the opening of the loading gate is necessary to load the template DNA. Myx is an antibiotic that binds to the switch 2 region, under the clamp, to prevent clamp closing/opening and inhibit transcription initiation. To study the inhibition mechanism of Myx in atomic resolution, we built quasi-Markov State Models (qMSM) based on extensive molecular dynamics (MD) simulations. qMSM can obtain the slow timescale dynamics of complex conformational change using shorter MD simulations compared to traditional MSM. qMSM identifies four states of clamp opening: closed, two partially closed, and open states. We have shown that the opening of the Myx binding site only occurs in a partially closed state, which allows binding of Myx. This suggests that Myx selectively binds to a partially closed state and follows the conformational selection mechanism. Notably, we find that β-lobe opening is sufficient for loading of DNA during transcription initiation. This highlights the critical role of β-lobe to initiate transcription and β-lobe's potential as a target for future antibiotics development. We also utilize qMSMto study the folding of the trigger loop (TL) of RNAP. During RNA elongation, TL closes/folds to stabilize the NTP substrate in the active site for catalysis. CBR is antibiotics that inhibit the folding of TL. Studying the molecular mechanism of TL folding will be useful to understand the inhibition mechanism of antibiotics, CBR, and future drug design targeting TL.