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Significance 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.

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