In the study published in the journal Nature Communications on April 15, entitled “A widely conserved protein Rof inhibits transcription termination factor Rho and promotes Salmonella virulence program”, Prof. WANG Chengyuan’s group at the Institute of Shanghai Institute of Immunity and Infection of the Chinese Academy of Sciences reported that Rof is a conserved antiterminator and the cryogenic electron microscopy structure of Rho-Rof antitermination complex. Further in vivo analyses in Salmonella Typhimurium show that Rof is required for virulence gene expression and host cell invasion, unveiling a novel physiological function of Rof and transcription termination in bacterial pathogenesis.

The Rho factor is a ring-shaped ATP-dependent hexameric helicase that is conserved throughout the bacterial kingdom. Rho plays an indispensable function in transcriptional termination and gene regulation in bacteria. Coordinated with transcription-translation coupling and mediated by the transcription elongation factor NusG, Rho modulates factor-dependent transcription termination and regulates the gene expression. Rho also separates transcription units, represses xenogenic genes, silences antisense RNAs, removes stalled RNAP from DNA thus maintaining genome stability, and prevents the formation of R-loops.

In the model bacterium Escherichia coli, about half of the transcription events are terminated by Rho factors. Five decades of genetic and biochemical experiments indicate that Rho-dependent transcription termination involves a series of transient steps. As the crucial first step, Rho recognizes a long C-rich RNA sequence (Rho utilization site; rut site) through its primary binding site (PBS) and binds with the rut site. Upon binding, Rho undergoes conformational changes from open-ring state to a catalytically competent, close-ring state. In the final step, Rho performs ATP-hydrolysis-dependent 5’-> 3’ translocation on mRNA, applying mechanical force to the transcription elongation complex (TEC) and triggering termination. Whereas the latter steps are facilitated by a number of structural and regulatory proteins such as NusG and NusA, whether and how the initial RNA binding step is regulated remains little understood.

Rof (Rho-off, also called YaeO) is the only E. coli host factor that directly interacts with Rho and inhibits the Rho-dependent transcription termination. However, whether Rof forms a protein complex with Rho and the basic nature of the interactions have been elusive. The regulatory and physiological functions of Rof as a conserved host factor in bacteria are completely unknown.

Herein, the researchers determined the atomic cryo-EM structure of Rho-Rof antitermination complex and revealed the molecular interactions between Rho and Rof.

Firstly, together with the recently determined Rho-dependent pre-termination complexes (Nature 2023), the new structure shows that Rof directly binds with Rho N-terminal domain (PBS site) and disrupts the interactions between PBS ligand RNA and Rho. Rof regulates the initiation of Rho-dependent termination and inhibits termination efficiency.

Furthermore, in vivo assays showed that Rof plays crucial functions in virulence regulation in the model bacterial pathogen Salmonella enterica serovar Typhimurium. Deletion of rof significantly reduced the expression of multiple virulence factors (hilA, prgH and sopB) that are required for Salmonella invasion of host cells.

Finally, the researchers suggest a model of Rof-Rho in virulence regulation in S. Typhimurium. Under OFF conditions, the pervasive transcription of SPI-1 genes (which contains AT-rich sequences) is silenced by both chromatin-binding protein H-NS and Rho, the latter of which recognizes rut site sequence and prevents unwanted transcription of virulence genes (Fig. 1, upper panel). During infection, Rof blocks Rho binding with rut site and inhibits Rho-dependent termination, leading to displacement of H-NS from SPI-1 locus, continued transcription of virulence genes, and successful host invasion (Fig. 1, lower panel). These results for the first time showed a physiological function of Rof in bacterial pathogens and in virulence regulation.