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 Delta S (+6.4 cal mol(-1) deg(-1)) and a negative Delta H (-11.3 kcal mol(-1)) combine to give a Delta G equal to -13.3 kcal mol(-1) at 37 degrees 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 (delta log K-a/delta log [NH4+] = +2.7 +/- 0.1). The dependence of K-d On the ionic strength and the positive Delta S suggests that hydrophobic and stacking interactions contribute significantly to the formation of the RNase P holoenzyme.