A Large Insertion Domain in the Rho Factor From a Low G plus C, Gram-negative Bacterium is Critical for RNA Binding and Transcription Termination Activity

Rho-dependent termination of transcription (RDTT) is a critical regulatory mechanism specific to bacteria. In a subset of species including most Actinobacteria and Bacteroidetes, the Rho factor contains a large, poorly conserved N-terminal insertion domain (NID) of cryptic function. To date, only two NID-bearing Rho factors from high G + C Actinobacteria have been thoroughly characterized. Both can trigger RDTT at promoter-proximal sites or with structurally constrained transcripts that are unsuitable for the archetypal, NID-less Rho factor of Escherichia coli ((Ec)Rho). Here, we provide the first biochemical characterization of a NID-bearing Rho factor from a low G + C bacterium. We show that Bacteroides fragilis Rho ((Bf)Rho) is a bona fide RNA-dependent NTPase motor able to unwind long RNA:DNA duplexes and to disrupt transcription complexes. The large NID (similar to 40% of total mass) strongly increases (Bf)Rho affinity for RNA, is strictly required for RDTT, but does not promote RDTT at promoter-proximal sites or with a structurally constrained transcript. Furthermore, the NID does not preclude modulation of RDTT by transcription factors NusA and NusG or by the Rho inhibitor bicyclomycin. Although the NID contains a prion-like Q/N-rich motif, it does not spontaneously trigger formation of beta-amyloids. Thus, despite its unusually large RNA binding domain, (Bf)Rho behaves more like the NID-less (Ec)Rho than NID-bearing counterparts from high G + C Actinobacteria. Our data highlight the evolutionary plasticity of Rho's N-terminal region and illustrate how RDTT is adapted to distinct genomic contents. (C) 2021 Elsevier Ltd. All rights reserved.

Automated summary

Rho-dependent termination of transcription (RDTT) is a critical regulatory mechanism specific to bacteria, with the Rho factor containing a large, poorly conserved N-terminal insertion domain (NID) in some species. In this study, the biochemical characterization of a NID-bearing Rho factor from a low G + C bacterium was provided, revealing its behavior more like the NID-less Rho factor. This highlights the evolutionary plasticity of Rho's N-terminal region and how RDTT is adapted to distinct genomic contents.