Fig. 5: Effect of Rof on RNA binding by ρ.
a Principles of fluorescence anisotropy assays used in (b) and (g). Isolated fluorescence-labeled (red star) dC15 DNA oligos (gray) or rU12 RNA oligos (gold) exhibit high tumbling rates (low fluorescence anisotropy). In the presence of ρ, dC15 binds to the PBSes of ρ, which reduces its tumbling rate (increased fluorescence anisotropy). In the presence of dC15, rU12 binds to the SBS of ρ, which leads to ρ ring closure. b PBS binding. Top, binding of dC15 to the ρ PBSes in the presence of increasing concentrations of ρ. Bottom, effect of Rof variants on the binding of dC15 to the PBSes. Data of two independent experiments and fits to a single-exponential Hill function (see Methods) are shown. Source data are provided as a Source Data file. c CryoEM reconstruction of the ρ-rut RNA structure. ρ forms a closed ring and is bound by RNA at the NTD of each protomer and at the SBS. Only RNA regions bound at the SBS and at the ρc NTD were modeled. RNA density and model, gold. As originally proposed2,5, ρ subunits are labeled in opposite directions around open and closed ρ rings (compare to Fig. 2b). We therefore labeled subunits of open ρ with capitals (A-F) and subunits of closed ρ with lower case letters (a–f). Rotation symbols in this and (d) views are relative to (c). d ρ interaction with RNA at the PBS. Six RNA residues (cartoon) fit to the density observed at ρc (semitransparent surface). e Path of the RNA along the PBS. ρ residues are shown as sticks and labeled. f Structural comparison between ρ-rut RNA and ρ6-ADP-Rof5 complexes. Structures of the complexes were superimposed based on the ρc/ρC subunits. Bound Rof sterically hinders RNA accommodation at the extended PBS. g Ring closure assays. Left, fluorescence anisotropy of 5’-FAM-labeled rU12 in the presence of increasing amounts of ρ. Right, Rof inhibits ρ ring closure. Data of two independent experiments and fits to a single-exponential Hill function (see Methods) are shown. Source data are provided as a Source Data file.
