Transcription, the process by which RNA polymerases synthesize RNA from a DNA template, is fundamental to life. RNA polymerases are able to synthesize very long RNAs, up to one million nucleotides in humans, but must do this in one try – any prematurely released message is lost. Each domain of life has evolved unique systems that support uninterrupted transcription of "good" genes while silencing "bad" ones. In bacteria, Rho (ρ), best characterized in Escherichia coli, triggers release of damaged or harmful RNAs from the transcribing RNA polymerase.

Rho, a ring shaped hexameric motor protein, is able to bind to and translocate along single-stranded RNA using NTP hydrolysis. It possesses two RNA-binding sites, the primary binding site (PBS) which runs around the outer rim, and the secondary binding site (SBS) within the ring’s center. Rho adopts two conformations: an inactive open ring (akin to the shape of a lock washer) that allows RNA to be loaded into the center of the ring, and an active closed ring that traps RNA inside the ring and activates Rho ATPase. Additionally, Rho can directly bind RNA polymerase and render it inactive.

Unrestrained Rho can target any RNA and is abundant during all stages of E. coli growth. During fast growth, the translating ribosomes shield mRNAs from Rho and rRNAs are protected by dedicated antitermination complexes; Rho then acts as a quality control sentinel that targets only useless RNAs. Nonetheless, it remains unclear how Rho activity is restrained during periods of stress or slow growth, when transcription is uncoupled from translation, yet many genes still need to be transcribed.

This paper investigates Rof (a.k.a YaeO), a cellular inhibitor of Rho whose detailed mechanism remained unknown for over 25 years after its discovery. Rof is a structural homolog of the RNA chaperone Hfq, a global regulator that affects mRNA stability, Rho-mediated termination, and translation. Said et al. used cryogenic electron microscopy (cryoEM) to obtain a structure of a Rho-Rof complex in which five Rof monomers bind at the interface between two Rho protomers of the hexamer. In this Rof₅:Rho₆ complex, both proteins undergo conformational changes that lock Rho in the open-ring conformation. Rof is bound near the PBS, supporting a model in which Rof competes with RNA, but does not directly block PBS.

The open-ring Rho-Rof structure prompted the authors to obtain a structure of the closed-ring Rho bound to a long RNA. In this structure, RNA occupies both the PBS and SBS and makes additional interactions with an "extended" PBS. The bound Rof would block latter contacts, as well as Rho interactions with the RNA polymerase, revealing a two-pronged approach to inhibition. Indeed, biochemical experiments confirmed that Rof competes with RNA and the transcription complex for binding to Rho and inhibits Rho-mediated termination in a purified in vitro system. These experiments also demonstrated the significance of the contacting residues in the structure, since binding is lost when they are mutated.

Additional in vivo assays, utilizing a growth defect phenotype, further validated the importance of the Rho-Rof binding residues in the structure. Overexpression of wild-type Rof inhibits E. coli growth, similar to the inhibition observed when Rho is inactivated by the drug bicyclomycin. In contrast, overexpression of single residue mutants of Rof had limited or no deleterious impact on growth, highlighting the importance of these residues in interacting with Rho.

In conclusion, this paper provides insight into the structural mechanism underlying Rof’s inactivation of Rho, suggesting its role in modulating Rho activity during periods of stress. Bacteria encounter a multitude of adverse conditions where inhibiting Rho may be crucial for survival in specific scenarios. These findings support the hypothesis that Rof is part of a group of specialized anti-termination factors that deactivate Rho across various stress conditions, promoting bacterial survival and recovery. Ongoing research is being done to elucidate the specific conditions to which Rof responds.