4Helmholtz-Zentrum Berlin für Materialien und Energie, Macromolecular Crystallography, Albert-Einstein-Straße 15, D-12489 Berlin, Germany
Most bacteria with sequenced genomes encode ρ, a hexameric RNA-dependent ATPase. In many of these species, ρ is essential. ρ is a paradigmatic transcription termination factor that defines the ends of 20-30 % of transcription units in Escherichia coli. Based on this capacity, ρ also mediates attenuation in 5’-untranslated regions, limits the extent of antisense transcription, silences foreign genes and safeguards genomes by restricting R-loops. ρ activity involves conformational transitions between an inactive open-spiral and an active closed-ring state, modulated, among others, by nucleotide and RNA binding. On RNA polymerase, ρ-dependent termination can be counteracted by various transcription factors [1], but only two known protein inhibitors target isolated ρ, including the polarity suppression protein, Psu, of pirate bacteriophage P4. Presently, the molecular basis of Psu-mediated ρ inhibition is unknown. We elucidated cryogenic electron microscopy structures of ρ-Psu complexes, in which multiple Psu dimers laterally clamp two ρ spirals, stabilizing them in an open conformation. Remarkably, Psu increases the helical pitch of the spirals, fostering their expansion by further ρ subunits up to the nonameric state. Consistent with Psu trapping ATP analogs at their binding sites on ρ, ATP stabilizes the ρ-Psu interaction, and Psu reduces ATP binding kinetics, thereby inhibiting the ρ ATPase. Also, fully consistent with the structures, Psu counteracts RNA binding at the center of the ρ spirals, which requires ring closure. Structure-guided exchange of ρ-Psu contact residues reduced complex formation, decreased Psu-mediated ρ ATPase inhibition and undermined Psu-mediated inhibition of ρ-dependent termination in vivo. Our findings reveal that Psu implements a unique mechanism – forced hyper-oligomerization – to inhibit ρ, which may inform the development of novel, ρ-targeting anti-microbials.
1. Said N, Hilal T, Sunday ND, Khatri A, Bürger J, Mielke T, Belogurov GA, Loll B, Sen R, Artsimovitch I, Wahl MC (2021) Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase ρ. Science 371, eabd1673.