The emergence of hyper-virulent and/or multi-drug-resistant bacteria calls for renewed efforts to understand the mechanisms that allow bacteria to evolve by incorporating and expressing foreign genes. Improving such understanding is an obligate step toward the development of alternative antibacterial strategies. Transcription termination factor Rho, an essential factor in many bacteria, mediates a large fraction (20-50%) of transcription termination events in E. coli and related species, and is thought to modulate expression of horizontally-acquired genes. Lying at the heart of bacterial expression programs, Rho constitutes an attractive therapeutic target for the development of new antibiotics. This ring-shaped RNA helicase targets transcriptional complexes and R-loops, its function being intimately linked to the specific flow of gene expression in bacteria where transcription and translation are coupled. Rho protein does not simply modulate intrinsic properties of the ribosome or RNA polymerase, as do other regulatory factors/signals, but adds layers of physical and dynamic complexity to the system by operating as an ATP-fueled, traveling molecular motor. Probably because of the level of sophistication of the functions and the mechanisms in which Rho participates, crucial elements of Rho biology remain incompletely understood.
The present proposal is based on a set of solid preliminary data, and years of successful collaboration, reinforcing our conviction that a multidisciplinary approach is the best strategy towards understanding how the action of Rho is integrated in the dynamics of the transcription elongation process. We plan to combine advanced genetics, quantitative imaging, combinatorial biochemistry, computational modeling and single-molecule detection and nanomanipulation methods in a comprehensive effort to gain further knowledge on all aspects of Rho function. In particular, “Ensemble” spectroscopic and single molecule experiments will reveal key facets of the mechanism and dynamics controlling the catalytic cycle of Rho and its interactions with the transcriptional machinery. Superresolution and fluctuation microscopies will explore the subcellular locations of Rho molecules and establish its spatial relationships with RNA polymerase, a point that remains unresolved and somewhat controversial. Functional/genomic SELEX approaches will allow identifying the RNA binding sites and termination signals used by Rho on a genome scale, including at loci repressed/silent under standard laboratory growth conditions. Bioinformatics and numerical simulation will decipher the rules linking Rho functional response to the context and organization of the termination signals. Through this integrated approach we will develop new experimental assays and predictive tools using Salmonella and E. coli as benchmark and model systems. We will then use them to perform a comparative study of Rho’s seminal and specific features in the phylodivergent, pathogenic microorganism Mycobacterium tuberculosis, a main objective being to understand the mechanism(s) of resistance of the M. tuberculosis Rho factor to Bicyclomycin, the only known antibiotic targeting Rho.
The consortium is constituted by three well-established research groups with internationally recognized expertise in their own research areas and fully active in the field of transcription termination/Rho research. The group of E. Margeat (coordinator, CBS, Montpellier) contributes extensive knowhow in biophysics, proving the expertise in single-molecule detection, advanced microscopies, and nanomanipulation methods. Marc Boudvillain (CBM, Orleans) and his group bring essential knowledge and expertise in combinatorial RNA biochemistry and enzymology and finally, Nara Figueroa-Bossi’s group (I2BC, Gif-sur-Yvette) provides long-standing experience with Salmonella, recombineering mutagenesis methods and bacterial genetics.
Monsieur Emmanuel Margeat (Centre de Biochimie Structurale)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
CBS Centre de Biochimie Structurale
CNRS-CBM Centre National de la Recherche Scientifique-Centre de Biophysique Moléculaire
I2BC Institut de Biologie Intégrative de la Cellule
Help of the ANR 540,000 euros
Beginning and duration of the scientific project: September 2015 - 48 Months