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

Emmanuel Saridakis,Josephine Lai-Kee-Him,Isabelle Simon,Kevin Martin,Emmanuel Margeat,Rishi Vishwakarma,Marc Boudvillain,Patrick Bron,Martin Cohen-Gonsaud,Franck Coste

doi:10.1038/s42003-022-03069-6

Abstract

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 [MtbRho] 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 MtbRho at 3.3 Å resolution. The MtbRho 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 MtbRho. 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 MtbRho and provides a framework for future antibiotic development.

Highlights

The bacterial Rho factor is a ring-shaped motor triggering genome-wide transcription termination and R-loop dissociation
We show that M. tuberculosis Rho (MtbRho) can adopt an open, ring-shaped hexamer conformation that mimics that observed for Escherichia coli Rho (EcRho)[18]
Since some particles display an open conformation and the intensity of the MtbRho subunits is not uniform in negative stain images, we surmise that this discrepancy stems from a combination of several parameters such as the collapse and flattening of particles due to their drying during sample preparation, a preferential orientation of the negatively stained particles onto the carbon film, and a misalignment of hexameric particles during the 2D classification

Summary

Introduction

The bacterial Rho factor is a ring-shaped motor triggering genome-wide transcription termination and R-loop dissociation. The analysis of the closed ring dataset did not permit to compute a reliable and interpretable map, while particles corresponding to the open conformation allowed us to compute a cryo-EM map at 3.3 Å resolution (which led to the structure of the hexamer in the open ring conformation), showing an open-ring organization of the complex resulting from the assembly of six MtbRho subunits

Results

Conclusion