Rho-dependent transcription termination: mechanisms and roles in bacterial fitness and adaptation to environmental changes

Historical model supported by extensive biochemical evidence (Jin et al. 1992; Steinmetz and Platt 1994; Richardson and Richardson 1996; Xu et al. 2002; Park and Roberts 2006; Schwartz et al. 2009; Kalyani et al. 2011; Rabhi et al. 2011b), single-molecule manipulation studies (Koslover et al. 2012; Gocheva et al. 2015), and structural analyses (Skordalakes and Berger 2003; Thomsen and Berger 2009; Lawson et al. 2018; Molodtsov et al. 2023).

Consistent with in vitro enzymatic properties of Rho, including RNA-stimulated ATP hydrolysis (Lowery-Goldhammer and Richardson 1974) and ATP-dependent, 5′→3′ directional translocation along RNA leading to disruption of RNA–DNA duplexes (Brennan et al. 1987; Walmacq et al. 2004, 2006) or biotin–streptavidin complexes (Schwartz et al. 2007).

Genetic evidence for kinetic coupling: Slow RNAP mutants suppress RDTT defects caused by rho and nusG mutations or by inhibitors such as Psu and Bycyclomycin (Shashni et al. 2012), supporting the model of kinetic coupling between Rho and RNAP translocations (Jin et al. 1992).

Cryo-EM reconstructions of pretermination complexes are compatible with this model (Molodtsov et al. 2023; Murayama et al. 2023); the nucleic acid substrates and assembly strategy used in these studies have been validated through biochemical RDTT assays.

Recently proposed model based on new evidence from a biochemical study (Epshtein et al. 2010) and cryo-EM reconstructions of pretermination complexes (Hao et al. 2021a; Said et al. 2021) containing NusA and NusG.

Colocalization of Rho and RNAP along the Escherichia coli genome revealed by ChIP-seq experiments (Mooney et al. 2009).

Parallel RDTT pathway(s) supported by single-molecule fluorescence studies (Song et al. 2022, 2023).

Cofactor missing in cryo-EM structures: E. coli “catch-up” pretermination complexes characterized by cryo-EM (Molodtsov et al. 2023) did not include NusA.

Biochemical validation missing: The reconstituted “stand-by” complexes on DNA bubble substrates (Hao et al. 2021a; Said et al. 2021) have not been shown to support RDTT.

Inactive Rho: In the “stand-by” complexes, the Rho hexamer adopts an open, catalytically inactive conformation and binds RNAP in an orientation incompatible with its translocase activity (Fig. 2D), suggesting the captured state may be off-pathway or represent an early, preproductive intermediate.

Off-pathway evidence: Biochemical characterization of analogous complexes from Thermus thermophilus supports an off-pathway interpretation (Murayama et al. 2023).

Uncertain RNA trajectory: The proposed inverted RNA contact sequence (SBS→PBS) (Hao et al. 2021a) lacks strong experimental validation and may instead reflect off-pathway RNA sequestration by NusA.

Steric hindrance at the PBS: Limited PBS accessibility in the “stand-by” complexes (Fig. 2D) raises questions about Rut site recognition, especially in cases of conditional RDTT requiring RNA structural remodeling.