Kinetic and Thermodynamic Analysis of RNA-Protein Interactions in the RNase P Holoenzyme from Escherichia coli

A gel retardation assay has been used to examine the kinetic and equilibrium properties of the interaction between C5 protein and M1 RNA in the formation of the ribonuclease P holoenzyme from Escherichia coli. The interaction is relatively insensitive to the identity of the monovalent anions present and to pH in the range 7.0–9.0, but it has a more critical requirement for specific monovalent and divalent cations: NH₄⁺, K⁺, Mg²⁺, Ca²⁺, and Mn²⁺ all promote efficient formation of the complex. A positive ΔS (+6.4 cal mol⁻¹ deg⁻¹) and a negative ΔH (-11.3 kcal mol⁻¹) combine to give a ΔG equal to -13.3 kcal mol⁻¹ at 37 °C in 0.42 M salt. The binding reaction is sensitive to the concentration of monovalent and divalent cations, with the affinity increasing with increasing ionic strength (δ log K_a/δ log [NH₄⁺] = +2.7 ± 0.1). The dependence of K_d on the ionic strength and the positive ΔS suggests that hydrophobic and stacking interactions contribute significantly to the formation of the RNase P holoenzyme.