Fig. 5: (p)ppGpp promotes reversible ρ hyper-oligomerization.

a Detection of ρ oligomers by BMOE crosslinking in the presence of ADP, GDP, and (p)ppGpp; protein marker sizes are indicated on the left. Experiments were performed three times independently with similar results. Source data are provided as a Source Data file. b Distribution of oligomeric states induced by different nucleotides; reported as means, n = 3; see Source data for quantification. c Structure of WT ρ bound to pppGpp (this study). ρ binds pppGpp at its nucleotide-binding pocket. The α5/α6 loop (residues 149–153 in orange) and helix α6 (residues 154–166 in orange) are indicated. Side chain residues important for nucleotide coordination are shown as sticks; compared to Fig. 1e–g. d 2 mM ADP- and ppGpp-stabilized ρ polymers were formed in vitro for 10 min and challenged with a non-hydrolyzable ATP analog (20 mM) for 30 min, followed by BMOE crosslinking. Percentages of ρ found in the D (dodecamer) and F (filament) fractions are shown as means ± SD; n = 4. Source data are provided as a Source Data file. e S84C ρ forms dodecamers in response to ppGpp. Experiments were performed three times independently with similar results; source data are provided as a Source Data file. f CryoEM map and structure of BMOE-crosslinked ρ^X1 variant in the presence of ppGpp; BMOE bridging two C84 residues is shown at the bottom. g Comparison of crosslinked ρ dodecamer to G150D^F-ADP. Two ρ hexamers in ρ^X1-BMOE structure are connected N-to-N. In contrast, in the G150D^F-ADP structure, ρ subunits are continuously joined C-to-N. All ρ variants used here are untagged.