title: Kinetic and thermodynamic analysis of RNA-protein interactions in the RNase P holoenzyme from Escherichia coli.
abstract: 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 Ka/delta log [NH4+] = +2.7 +/- 0.1). The dependence of Kd 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.
authors.affiliation: Department of Biology, Yale University, New Haven, Connecticut 06511.
authors.forename: S J
authors.initials: SJ
authors.lastname: Talbot
authors.forename: S
authors.initials: S
authors.lastname: Altman
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002413:Cations, Divalent; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D016337:RNA, Catalytic / Q000378:metabolism*; D043262:Ribonuclease P; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D002413:Cations, Divalent; D029968:Escherichia coli Proteins; D016337:RNA, Catalytic; D012996:Solutions; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli