Bacterial RNA Polymerase (RNAP) is an essential enzyme that transcribes the RNA from DNA. RNAP has been the antibiotics target due to its vital role in the bacterial life cycle. Yet, the emergence of antibiotic resistance urges the development of new antibiotics. RNAP requires the conformational changes of several structural modules to perform its function. 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. This is followed by closing the loading gate to stabilize the RNAP and DNA complex. Myxopyronin (Myx) is an antibiotic targeting the movement of the clamp domain to inhibit transcription initiation. To study the recognition mechanism of Myx, we constructed quasi-Markov State Models (qMSM) based on extensive molecular dynamics (MD) simulations. We found that the clamp interconverts between four states: closed, two partially closed, and open states. Yet, Myx can only bind to a partially closed state, which suggests that the binding of Myx follows the conformational selection mechanism. Importantly, we found that the opening of β-lobe only is sufficient for the loading of DNA, highlighting the critical role of β-lobe and its potential for future antibiotics development. qMSM is a great tool to obtain the slow timescale dynamics of complex conformational change using shorter MD simulations compared to conventional MSM. It is also useful to analyze the recognition mechanism of antibiotics to the target protein. Thus, we will also use qMSM to study the trigger loop (TL) domain of bacterial RNAP. Folding of TL is crucial to stabilize the NTP substrate in the active site for the addition of NTP to the RNA chain. CBR is a class of antibiotics that has been shown to inhibit the folding of TL. Studying how TL folds will be important to understand the recognition mechanism of CBR, as well as future antibiotics design targeting TL.

Elucidating the inhibition mechanism of antibiotics in bacterial RNA polymerase using quasi-markov state models

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