VI. RNA AND CODING PROBLEMS 281 



conclusion that the RNA synthesized is a true complement of the 

 primer. This enzyme, tlicn, provides a means for the synthesis of DNA- 

 like or messenger RNA in biologic systems. 



VI. RNA Involvement in in Vitro Protein Synthesis 



A. REQUIREMENT FOR DNA-DIRECTED RNA SYNTHESIS 



Probably the first evidence for a synthetic pathway for RNA via 

 nucleoside triphosphates, and, at the same time, the first indication that 

 this RNA may be required for protein synthesis, was reported by 

 Spiegelman (1959) with cell-free fractions from microbial systems. 

 Spiegelman found the protoplast membrane fraction highly potent in 

 leucine-C^'*-incorporating ability into acid-insoluble protein, exceeding 

 by a factor of 100 that found in the high-speed pellet or supernatant 

 fraction. There was a definite requirement for all four nucleoside tri- 

 phosphates, the presence of these compounds eliminating a lag in incor- 

 poration, and they could not be substituted for by the corresponding 

 diphosphates. Nisman and Fukuhara (1959) and Nisman et al. (1961), 

 likewise employing the E. coli membrane fraction, were able to assign 

 a definite importance to the presence of intact DNA in the cell-free 

 synthesis of /3-galactosidase and the incorporation of amino acids into 

 protein. A clear-cut approach to the question of the necessity for DNA 

 was carried out by Novelli and his co-workers (1961). By the device of 

 either UV-irradiation of the supernatant fraction or X-radiation of 

 whole cells, they demonstrated an absolute requirement for the addition 

 of DNA to their cell-free system (particles + supernatant) for the syn- 

 thesis of /3-galactosidase. Moreover, only DNA prepared from cells 

 containing the gene for j8-galactosidase was effective. The system re- 

 quired all for ribonucleoside triphosphates for enzyme synthesis and the 

 authors invoked a mechanism for the synthesis of RNA as an inter- 

 mediate, or messenger, in the transmission of information from the 

 specific DNA to the enzyme-fomiing system. 



Tissieres et al. (1960) claim that the major particle site for protein 

 synthesis in bacterial systems is the 70S ribosomes. Tissieres has shown 

 (Tissieres and Hopkins, 1961a,b) that the RNA synthesized in vitro 

 from the triphosphates has similar sedimentation properties in a sucrose 

 gradient as that observed by Gros et al. (1961) following pulse labeling 

 in vivo. In addition, this RNA seems to be required for the amino acid 

 incorporation observed in the 70S ribosomes (Tissieres and Hopkins, 

 1961b). 



