title: Direct observation of the translocation mechanism of transcription termination factor Rho.
abstract: Rho is a ring-shaped, ATP-fueled motor essential for remodeling transcriptional complexes and R-loops in bacteria. Despite years of research on this fundamental model helicase, key aspects of its mechanism of translocation remain largely unknown. Here, we used single-molecule manipulation and fluorescence methods to directly monitor the dynamics of RNA translocation by Rho. We show that the efficiency of Rho activation is strongly dependent on the force applied on the RNA but that, once active, Rho is able to translocate against a large opposing force (at least 7 pN) by a mechanism involving 'tethered tracking'. Importantly, the ability to directly measure dynamics at the single-molecule level allowed us to determine essential motor properties of Rho. Hence, Rho translocates at a rate of ∼56 nt per second under our experimental conditions, which is 2-5 times faster than velocities measured for RNA polymerase under similar conditions. Moreover, the processivity of Rho (∼62 nt at a 7 pN opposing force) is large enough for Rho to reach termination sites without dissociating from its RNA loading site, potentially increasing the efficiency of transcription termination. Our findings unambiguously establish 'tethered tracking' as the main pathway for Rho translocation, support 'kinetic coupling' between Rho and RNA polymerase during Rho-dependent termination, and suggest that forces applied on the nascent RNA transcript by cellular substructures could have important implications for the regulation of transcription and its coupling to translation in vivo.
authors: CNRS UMR5048, Centre de Biochimie Structurale, 29 rue de Navacelles, 34090 Montpellier, France INSERM U554, 34090 Montpellier, France Universités Montpellier 1 et 2, 34090 Montpellier, France.
authors: Veronika
authors: V
authors: Gocheva
authors: CNRS UMR5048, Centre de Biochimie Structurale, 29 rue de Navacelles, 34090 Montpellier, France INSERM U554, 34090 Montpellier, France Universités Montpellier 1 et 2, 34090 Montpellier, France.
authors: Antoine
authors: A
authors: Le Gall
authors: CNRS, Centre de Biophysique Moléculaire, rue Charles Sadron, 45071 Orléans, France ITP Sciences Biologiques & Chimie du Vivant, Université d'Orléans, France.
authors: Marc
authors: M
authors: Boudvillain
authors: CNRS UMR5048, Centre de Biochimie Structurale, 29 rue de Navacelles, 34090 Montpellier, France INSERM U554, 34090 Montpellier, France Universités Montpellier 1 et 2, 34090 Montpellier, France marcelo.nollmann@cbs.cnrs.fr.
authors: Emmanuel
authors: E
authors: Margeat
authors: CNRS UMR5048, Centre de Biochimie Structurale, 29 rue de Navacelles, 34090 Montpellier, France INSERM U554, 34090 Montpellier, France Universités Montpellier 1 et 2, 34090 Montpellier, France marcelo.nollmann@cbs.cnrs.fr.
authors: Marcelo
authors: M
authors: Nollmann
mesh_terms: D007700:Kinetics; D008958:Models, Molecular; D021381:Protein Transport; D012313:RNA / Q000378:metabolism; D012234:Rho Factor / Q000737:chemistry / Q000378:metabolism*; D061806:Transcription Termination, Genetic
chemical_list: D012234:Rho Factor; D012313:RNA