280 ELLIOT VOLKIN 



V. In Vitro Synthesis of RNA 



A. POLYNUCLEOTIDE PHOSPHORYLASE 



In 1955 (ii'uiilx'i'^-Mana^o and Ochoa (lisc()\H'i(Ml an enzyme, jjolynu- 

 cleotidc iihosphoiylase. that fur the fii\><t time allowed the eell-free, 

 enzymatic synthesis of polyriboniu'leotide. Ribonucleoside diphosphates 

 serve as substrates in the reaction and the composition of the product 

 formed can be manipulated according to the relative concentrations of 

 the individual diphosphates. Thus, polynucleotides can be synthesized 

 ranging from the homopolymers to those containing all four bases in a 

 variety of ratios (Ochoa and Heppel, 1957). It would be difficult to 

 imagine that this mode of enzymatic synthesis is responsible for the 

 cellular production of messenger RNA. Thus, if rigorous biologic speci- 

 ficity is associated with the RNA, and this specificity is a function of the 

 nucleotide sequence, it seems unlikely that such sequences should depend 

 on the relative pool sizes of the nucleoside diphosphate precursors. In 

 view of the truly outstanding nature of the discovery of this enzyme, 

 however, it is pleasing to note the eventual great usefulness of this 

 system in the elucidation of the coding problem (see below). 



B. RNA POLYMERASE 



The independent discovery from the laboratories of Weiss and 

 Gladstone (1959), Hurwitz et nl. (1960), and Stevens (1960) of the 

 enzyme RNA polymerase has opened new avenues of approach not only 

 to the question of the biologic synthesis of RNA but also to the entire 

 question of specificity relations between RNA and protein. The enzyme 

 has been detected in a variety of mammalian (Weiss and Gladstone, 

 1959; AVciss, 1962) and plant (Huang et al, 1960) tissues, as well as in 

 bacteria (Hurwitz et a/., 1960; Stevens, 1960; Ochoa ef ai, 1961). RNA 

 polymerase, like DNA polymerase (Romberg et al., 1956; Bessman 

 et al, 1958), requires DNA as a primer, and utilizes the ribonucleoside 

 triphosphates as substrates. A net synthesis of the product can be 

 attained. Most importantly, the composition of the DNA primer deter- 

 mines the composition of the RNA product. All four nucleoside triphos- 

 phates must be present for RNA synthesis, although ATP can be 

 polymerized to polyadenylic acid (see also Spiegelman, 1959). The fact 

 that heat-denatured DNA (Geiduschck et al., 1961 ; Chamberlain and 

 Berg, 1962) and single-stranded DNA of (/)X174 phage (Chamberlain and 

 Berg, 1962) can serve as i)rimers, i)lus the decisions based on nearest- 

 neighbor frequency analysis (Weiss and Nakamoto, 1961), point to the 



