Fig. 4: ρ oligomers form and disperse in response to cellular cues.

a Sucrose density gradient analysis of WT and G150D ρ proteins in vitro (top) and in cells (bottom). ρ was detected by Western blotting with polyclonal anti-ρ antibodies. Tentative H, D, and F positions were assigned based on the sedimentation of known proteins. Experiments were performed three times independently with similar results. Source data are provided as a Source Data file. b WT, G150D, and ΩIS2 rho (ρ ↓ ) cells were streaked on LB plates with or without MUP. Experiments were performed three times independently with similar results. c–e Quantification of in vivo pelleting assays (Supplementary Fig. 10a). The fraction of ρ pelleted at 110,000 x g (representing oligomers) was determined relative to the total amount of ρ in the sample (pellet + supernatant) using Western blotting. c ρ forms oligomers following a 30 min exposure to antibiotics that inhibit protein or RNA synthesis. ─, none; NAL, nalidixic acid; MUP, mupirocin; RET, retapamulin; RIF, rifampicin. The p-value was calculated between (─, NAL) and (MUP, RET, RIF). d MUP effect in the ppGpp⁰ strain. e To investigate whether ρ oligomerization was reversible, cells were washed with LB following MUP treatment (carried out as in panel c) and incubated for 40 min in the absence of the antibiotic; control experiments demonstrated that cell growth did not resume after the 40 min recovery period (Supplementary Fig. 10c). All ρ variants used here are untagged. Two-tailed T test assuming unequal variance was used to determine p-values. Source data are provided as a Source Data file.