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

Cryo-EM shows that M. tuberculosis Rho-factor adopts an open, ring-shaped hexamer conformation and a steric bulk in the cavity for bicyclomycin binding, which explains resistance to the antibiotic. 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 angstrom 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 gamma-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.

Automated summary

Cryo-EM structure determination reveals that M. tuberculosis Rho-factor adopts an open, ring-shaped hexamer conformation and exhibits steric bulk in the cavity for bicyclomycin binding, leading to resistance to the antibiotic. The study also uncovers a leucine-to-methionine substitution that creates a steric bulk in the binding cavities, resulting in resistance to bicyclomycin at the expense of motor efficiency. This work contributes to understanding the unique features of M. tuberculosis Rho and provides insights for future antibiotic development.