Fig. 1: Rof structure, functions, conservation and genetic organization.

a Structural comparison of E. coli Rof (left) and Hfq (right). Proteins are shown in rainbow colors from N-termini (N; blue) to C-termini (C; red). b Cells transformed with plasmids expressing wild-type (wt) Rof and Rof variants under the control of an IPTG-inducible P_trc promoter or an empty vector were grown overnight in LB supplemented with carbenicillin at 32 °C. Serial 10-fold dilutions were plated on LB-carbenicillin in the presence of IPTG and incubated overnight at 32 °C. A set representative of five independent experiments is shown. Shine-Dalgarno (SD) elements are indicated by blue bars. c Rof inhibits ρ-dependent termination in vitro. Top, DNA templates encoding the rut⁺ λ tR1 (left) or rut^- siiE leader region (right) downstream of the λ P_R promoter and a C-less cassette that allows for the formation of synchronized, radiolabeled ECs halted 26 nts downstream of the transcription start site. Bottom, representative single-round transcription reactions. Halted ECs were incubated with ρ and/or Rof (where indicated), restarted by addition of NTPs, and subsequently quenched. Reactions were analyzed on 5% urea-acrylamide gels. Positions of the full-length run-off (RO) RNA and termination regions are indicated, as are the sizes of the molecular weight markers generated by γ³²P-labeling of pBR322 MspI fragments. The fractions of run-off RNA represent the mean ± SD of three independent experiments. Source data are provided as a Source Data file. d Rof distribution on the phylogenetic tree of Pseudomonadota. Each leaf represents one family. The height of the bar indicates the fraction of Rof in each family, with the highest one being 80%. e E. coli yae locus (MG1655 genome coordinates 213,630-216,140); the length of each ORF is indicated below the sequence, the nlpE gene is interrupted to save space. The arfB and rof genes lack SD elements and overlap with the respective upstream ORFs, yaeP and yaeQ, by 4 and 8 nts. The “insulating” REP elements are shown by dark gray boxes.