Enzymatic Synthesis of Ribonucleic Acid 1. PREPARATION OF RIBONUCLEIC ACID POLYMERASE FROM EXTRACTS OF MICROCOCCUS LYSODEIKTICUP

The problem of complex polynucleotide synthesis has received considerable attention from numerous investigators (l-3). Grunberg-Manago and Ochoa (4) and Kornberg, Lehman, and Simms (5) first successfully demonstrated the polymerization in vitro of riboand deoxynucleotides with cell-free extracts from microorganisms. Subsequent studies on the synthesis of ribonucleic acid and deoxyribonucleic acid in other laboratories, including our own, were guided primarily by the experience of these two groups. The different mechanisms and substrate specificities demonstrated for polynucleotide phosphorylase (6) and DNA polymerase (7) first directed our attention to the possible existence of yet other enzymes which catalyzed nucleotide polymerization. At that time, no adequate mechanism was known for the synthesis of RNA molecules with base compositions resembling that of DNA (8). A preliminary report from this laboratory (9) first described the presence of an enzyme in rat liver nuclei which carried out the incorporation of ribonucleotides into RNA and required the participation of all four ribonucleoside triphosphates. The properties of this mammalian cell-free particulate system for ribonucleotide incorporation were distinctly different from previously reported reactions (10). However, some investigators had described the stimulation of RNA synthesis by ribonucleoside diand triphosphates in cell membrane fractions prepared from Escherkhiu coli protoplasts (11). A more detailed analysis of the product formed by the liver nuclei system showed that ribonucleotide incorporation occurred throughout the entire RNA molecule and was dependent upon intact DNA (12). Subsequent reports have now confirmed the presence of similar reactions in extracts from other mammalian tissues (13), from plants (14), and from microorganisms (15-18). In all these cases, the reaction mechanism for the DNA-dependent synthesis of RNA (Fig. 1) appeared similar to the DNA-dependent synthesis of DNA (7). For this reason, we initially referred to this enzyme as RNA polymerase. More recently, we have found that bacterial RNA polymerase preparations also catalyze an RNA-dependent synthesis of RNA in a manner similar to the DNA-primed reaction (19). Originally we were unable to deter-