45] The use of membrane filtration to determine apparent association constants for ribosomal protein-RNA complex formation

Abstract

The chapter discusses the use of nitrocellulose membrane filtration that allows accurate measurement of the selective association of ribosomal proteins with RNA without disturbing the equilibrium. The ribosomal proteins are adsorbed, at appropriately low concentrations, to nitrocellulose membranes under conditions where specific protein-RNA complexes fail to be bound by the membrane. This observation offers the opportunity to separate rapidly unbound, or free protein from the protein involved in a protein- RNA complex. This method permits the determination of the amount of protein bound to RNA and the amount of free protein in reaction mixtures containing different relative concentrations of protein and RNA. The resultant data is used to calculate the number of binding sites for the protein on the RNA and the apparent association constant for the binding reaction. Protein-RNA complexes involving up to 12 different proteins and 16 S RNA readily passes through the membrane in RB solution. Thus, quantitative studies on protein binding to preformed protein-RNA complexes can be performed by this method. Other applications include the examination of different solvent conditions and their effect on the strength of the association. Besides being applied to the study of ribosome structure, this fractionation technique is adapted for use with other protein-nucleic acid systems.