Fig. 3: ADP-stabilized filamentation compromises the activity of ρ.

a The ADP bound ρ G150D•H₈ filament (F-ADP) and untagged WT-ADP structures show a strong cation-π interaction between R353 and W381, whereas the interaction seems to be more flexible in the untagged WT-ATP structure (PDB ID: 6WA8). Here, the density for R353 is rather weak at several protomer interfaces, indicating that the R353 side chain conformation is not stabilized by W381. Experimental density is in semitransparent surface representation. b Effects of filamentation on ATPase activity. 1.5 μM untagged ρ was incubated with 2.5 mM nucleotides (GDP/ADP) or water (none) for 10 min at room temperature to facilitate filament formation. Then rut site-containing RNA (see “Methods”) and ATP were added to 1.5 μM and 2 mM (containing 0.2 μCi/μL γ³²P-ATP) final concentrations, respectively. The reaction proceeded for 5 min before quenching by EDTA and products were resolved by TLC. The fraction of P_i was used as an indicator of ATPase activity. c Effects of filamentation on ρ-dependent termination. Nucleotide-stabilized filaments were formed by incubating 2.5 μM untagged ρ with 5 mM nucleotides (GDP/ADP) or water (none) for 10 min at room temperature. Then, the ρ-nucleotide mixture was diluted 5 times into halted radiolabeled transcription elongation complexes formed on the λ tR1 template (see “Methods”), and 150 μM NTPs were added to restart elongation. Positions of run-off (RO) transcript and ρ-dependent termination region are indicated on a representative urea-acrylamide gel. nts, nucleotides. The fraction of RO RNA was used to evaluate the termination activity of ρ. In vitro experiments were performed three times independently with similar results. Two-tailed T test assuming unequal variance was used to calculate p-values. Source data are provided as a Source Data file.