37. NUCLEIC ACID AND PROTEIN SYNTHESIS 379 



terminal attachment of CMP is a reversible pyrophosphate splitting reac- 

 tion: pyrophosphate inhibits the forward reaction and PP 32 is incorporated 

 into CTP in the back reaction only in the presence of sRNA. Herbert 

 has demonstrated directly that PP accumulates as the product of the 

 terminal attachment of the AMP moiety of ATP to sRNA. 123 



As these workers have pointed out, it is clear that these terminal ad- 

 dition reactions are distinct from the reactions catalyzed by nucleoside 

 phosphorylase not only in that triphosphates are the immediate precursors 

 but also in that the attachment is specific for ATP and CTP and to a 

 lesser extent UTP. The terminal configuration is not altered by the pres- 

 ence of other nucleotide precursors. 



(4) The Enzymes Catalyzing the Reactions. Although the enzyme system 

 from the rat liver supernatant fraction catalyzing the attachment of ter- 

 minal nucleotides to sRNA has been studied by many workers, only one 

 report has been concerned with its purification. Herbert has had success 

 in this regard, using ammonium sulfate fractionation and adsorption on 

 a hydroxy apatite column. 123 The extent of purification of activity for 

 both CMP and AMP attachment is parallel at each step, suggesting that 

 a single enzyme may be involved for both nucleotide additions. Further- 

 more, the pH optima for attachment of both nucleotides are identical. 

 The system is sensitive to trypsin and to heating at 85°C. for a brief 

 period. The fractionation procedure also effects a complete separation of 

 these enzymes from amino acid activating enzymes, thereby permitting 

 the demonstration that the terminal AMP is derived directly from ATP 

 and not from the AMP moiety of the amino acyl adenylate. This point 

 will be discussed further below. 



(5) The Mechanism of the Terminal Nucleotide Attachment. The evidence 

 available thus far suggests that the reaction may be formulated in the 

 three steps depicted in Fig. 2. 



This formulation states that three known nucleotides are attached to 

 the end of a polynucleotide chain of unknown base sequence. (The two 

 last nucleotides, X and Y are shown). The two CMP moieties of two mole- 

 cules of CTP are first attached sequentially to the 3'-hydroxyl of the 

 ribose of the terminal nucleotide in the chain. Then the resulting terminal 

 CMP is covered by an AMP by a similar pyrophosphoryl cleavage of 

 ATP. The reaction is then complete and proceeds no further. The following 

 evidence supports this formulation 122 ' 123, 154 ' 155 : (1) incubation of sRNA 

 with CTP-C 14 results in the incorporation of C 14 into the RNA, and upon 

 alkaline hydrolysis approximately 50 % of the C 14 is recovered as cytidine, 

 and 50% as 2 / ,3'-cytidylic acid. (With the purified enzyme system of 

 Herbert, the results differ: almost all of the incorporated CMP-C 14 is 

 terminal. The formulation presented here is based on the observations 



