Purification and Properties of Bacteriophage f2 Replicase

The present dissertation concerns the purification and properties of an RNA-dependent ribonucleotide-polymerizing enzyme (RNA replicase) produced during infection of E. coli with the RNA bacteriophage f2. Studies on the RNA replicase of the distantly-related bacteriophage Q3 have established the feasibility of using a simplified assay for replicase activity based on the ability of the enzyme to polymerize GTP in the presence of polycytidylic acid template. A similar poly Cdependent poly G polymerase activity is detectable in E. coli infected with bacteriophage f2. The f2 poly G polymerase has been purified by ion exchange chromatography on DEAE cellulose, affinity chromatography on RNA cellulose and density gradient centrifugation. Highly purified preparations of phage-induced poly G polymerase consist predominantly of four proteins having approximate molecular weights of 75,000, 63,000, 46,000 and 33,000. The 63,000 m.w. protein has been identified as the phage-coded replicase protein based on co-electrophoresis of this polypeptide with replicase protein obtained from phage-infected cells. Partially purified preparations of f2 poly G polymerase exhibit replicase activity. Such preparations catalyze nucleotide polymerization in the presence of single-stranded f2 phage RNA or f2 complementary strand RNA, but are inactive with other viral and bacterial RNAs. The product of the in vitro enzymatic reaction has been analysed by RNA annealing techniques and found to consist entirely of polynucleotides homologous to f2 RNAs. In the presence of single-stranded phage RNA template, the enzyme synthesizes both complementary strand RNA and some product copies of the input template strand. However, the enzyme does not synthesize RNA in excess of the input template amount and is able to release only a small proportion of the product RNA as single-stranded viral RNA. Most of the product RNA remains in partially double-stranded complexes with template RNA. Higihly purified f2 poly G polymerase shows no replicase activity. It has been found that a factor fraction necessary for replicase activity separates from the poly G polymerase during glycerol gradient centrifugation. This fraction, which shows no nucleotide-polymerizing activity and is presumably of bacterial origin, restores replicase activity with both f2 single-stranded viral RNA and f2 complementary strand RNA to the 4-polypeptide poly G polymerase. The f2 replicase and poly G polymerase activities are quite similar with respect to such parameters as stability, temperature optimum, divalent cation requirements, phosphate insensitivity and template saturation kinetics. Template competition experiments further suggest that the synthetic polymer and phage RNA compete for binding to the enzyme. The effect of the ionic strength of the incubation medium on the two activities is quite different, however. Replicase activity is stimulated at ionic strengths up to about 0.1, while the poly G polymerase is markedly inhibited, even at quite low salt concentrations. The results of substrate saturation studies suggest that the affinity of replicase is considerably higher for ATP, CTP and UTP than for the chain-initiator nucleotide GTP. Furthermore, the complex saturation kinetics observed for GTP in both replicase and poly G polymerase reactions indicate the simultaneous interaction of more than one molecule of GTP with the enzyme. On the basis of the present studies with the f2 enz3rme and the considerable literature on other nucleotidepolymerizing enzymes, it is proposed that the replicase has separate active sites for RNA chain initiation and polymerization. It is postulated that the synthetic polymer-dependent reaction occurs primarily or exclusively at the chain initiation site, whose normal function is to recognize the 3\u27 terminus of phage RNA templates and carry out the polymerization of the 5\u27 terminal guanylate residues of phage RNAs. It is further suggested that replication of phage RNAs occurs primarily at the polymerization site, whose activity is governed by specific secondary interactions of the enzyme with natural template RNAs