Gel Retardation Analysis of the Interaction between C5 Protein and M1 RNA in the Formation of the Ribonuclease P Holoenzyme from Escherichia coli

Abstract

C5 protein binds specifically and with high affinity to M1 RNA to form the ribonuclease P holoenzyme of Escherichia coli. The interactions between the two subunits of the enzyme have been studied in vitro by a gel retardation assay. The stoichiometry of the subunits in the holoenzyme is 1:1. The dissociation constant (Kd) for the specific interactions of the subunits in the holoenzyme complex is < or = 0.4 nM. C5 protein also has nonspecific affinity for M1 RNA and a variety of other RNA molecules with Kd values in the order of 10-40 nM. Scatchard analysis of binding data suggests the existence of two modes of interaction between C5 protein and M1 RNA--one high-affinity and one low-affinity mode. Regions of M1 RNA essential for formation of the specific complex with C5 protein have been defined by deletion analysis and footprinting methods. Our data show that regions of M1 RNA that interact with C5 protein are clustered into three main areas that are localized between nucleotides 41-99, 168-198, and 266-287.