R17 RNA Replicase: Separation of Phage Coded and Host Proteins*

(1) Further purification of R17 RNA-dependent RNA polymerase (RNA replicase) of the first DEAE-cellulose fraction resulted in a complete loss of replicase activity, due to the dissociation of replicase into two protein components. This dissociation was effected by treating the enzyme fraction with a high salt concentration, followed by a second DEAE chromatography. One component is eluted from the column at 0.1 M KC1 (ø-factor) and the other at 0.24 M (Fraction DR) which contains functional DNA-dependent RNA polymerase (RNA polymerase, EC 2.7.7.6). The ø-factor does not possess enzymatic activity by itself. (2) Reconstitution of Fraction DR and ø-factor resulted in the restoration of RNA replicase activity and in the loss of RNA polymerase activity. (3) ø-factor was not present in non-infected E. coli, K12, Hfr strain; E. Coli, K12, F-strain; and E. coli B. (4) Fraction DR, on the other hand, could be isolated from these three strains of E. coli without infection. This fraction, which could be derived even from the female strain and contained RNA polymerase activity, catalyzed R17 RNA-directed RNA polymerization in the presence of the ø-factor. From these facts, it was concluded that the ø-factor is coded for by the phage genome and Fraction DR is bacterial proteins, and that the phage specific protein (ø-factor) has a close relationship to the host DNA-dependent RNA polymerase or its subunit(s).