GEL RETARDATION ANALYSIS OF THE INTERACTION BETWEEN C5 PROTEIN AND M1RNA IN THE FORMATION OF THE RIBONUCLEASE-P HOLOENZYME FROM ESCHERICHIA-COLI

C5 protein binds specifically and with high affinity to M1 RNA to form the ribonuclease P holoenzyme of Escherichia coli. The interactions b etween the two subunits of the enzyme have been studied in vitro by a gel retardation assay. The stoichiometry of the subunits in the holoen zyme is 1:1. The dissociation constant (K-d) for the specific interact ions of the subunits in the holoenzyme complex is less than or equal t o 0.4 nM. C5 protein also has nonspecific affinity for M1 RNA and a va riety of other RNA molecules with K-d values in the order of 10-40 nM. Scatchard analysis of binding data suggests the existence of two mode s of interaction between C5 protein and M1 RNA-one high-affinity and o ne low-affinity mode. Regions of M1 RNA essential for formation df the specific complex with C5 protein have been defined by deletion analys is and footprinting methods. Our. data show that regions of M1 RNA tha t interact with C5 protein are clustered into three main areas that ar e localized between nucleotides 41-99, 168-198, and 266-287.