Cryo-EM structure of transcription termination factor Rho from Mycobacterium tuberculosis reveals bicyclomycin resistance mechanism

The bacterial Rho factor is a ring-shaped motor triggering genome-wide transcription termination and R-loop dissociation. Rho is essential in many species, including in Mycobacterium tuberculosis where rho gene inactivation leads to rapid death. Yet, the M. tuberculosis Rho [(Mtb)Rho] factor displays poor NTPase and helicase activities, and resistance to the natural Rho inhibitor bicyclomycin [BCM] that remain unexplained. To address these issues, we solved the cryo-EM structure of (Mtb)Rho at 3.3 Å resolution. The (Mtb)Rho hexamer is poised into a pre-catalytic, open-ring state wherein specific contacts stabilize ATP in intersubunit ATPase pockets, thereby explaining the cofactor preference of (Mtb)Rho. We reveal a leucine-to-methionine substitution that creates a steric bulk in BCM binding cavities near the positions of ATP γ-phosphates, and confers resistance to BCM at the expense of motor efficiency. Our work contributes to explain the unusual features of (Mtb)Rho and provides a framework for future antibiotic development.