CE44 - Biochimie et chimie du vivant

An integrated view on the mycobacterial Rho-dependent transcription termination (RDTT) machinery: groundwork for future pathogen-specific therapeutics – MYCRHO

Submission summary

Transcription termination mediated by the ATP/RNA-dependent motor protein Rho (RDTT) is an essential regulatory mechanism in many bacteria. RDTT is specific to bacteria and thus constitutes an attractive target for the development of antibacterial prophylactic/therapeutic strategies. Our understanding of RDTT is based on studies of the Escherichia coli (Ec) paradigm but growing evidence suggests that RDTT mechanisms are markedly different in divergent species such as Mycobacterium tuberculosis (Mtb), the agent of human tuberculosis. For example, key interacting motifs in Rho, RNA polymerase, or RDTT co-factors from Ec are not conserved in Mtb. Moreover, Mtb-Rho is refractory to bicyclomycin, the only-known Rho inhibitor, and it harbors a large, taxa-specific insertion domain that is thought to govern its RNA target selectivity.
In the MYCRHO project, we will combine unprecedented biochemical probing of Mtb-Rho enzymatic activity genome-wide with state-of-the-art structural, microbiological, and proteomics approaches to build a comprehensive model of RDTT in the Mtb pathogen. First, we will use a new proprietary combinatorial procedure, H-SELEX, together with mass spectrometry proteomics to define the functional interactome and substrate repertoire of Mtb RDTT. Then, we will use NMR and cryoEM to explore the structural organization and molecular dynamics of the Mtb transcription complex along the Rho-dependent termination pathway. Finally, we will implement the first genuine biochemical and microbiological assays of Mtb RDTT to test salient features uncovered in previous tasks and validate, in vitro and in vivo, the molecular specifics of the Mtb RDTT machinery.
Through this multidisciplinary work, we will provide a new paradigm for RDTT in a pathogen, thereby facilitating future drug development and illuminating the evolutionary driving forces imposed on this critical regulatory mechanism across bacteria.

Project coordination

Marc Boudvillain (Centre national de la recherche scientifique)

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.

Partner

ICSN Centre national de la recherche scientifique
Washington University, Division of Biology and Biomedical Sciences
IRIM Centre national de la recherche scientifique
IGBMC Institut de Génétique et de Biologie Moléculaire et Cellulaire
CBM Centre national de la recherche scientifique

Help of the ANR 686,475 euros
Beginning and duration of the scientific project: January 2023 - 54 Months

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