I20 E. S. CANELLAKIS AND EDWARD HERBERT 



such a radioactive S-RNA preparation is added to an incubation medium 

 containing the ribonucleotide-incorporating enzymes, and varying con- 

 centrations of Mg + + and pyrophosphate, a progressive loss of radioactivity 

 occurs, indicating the removal of the terminal ribonucleotides in the S-RNA 

 molecule. 



Since pyrophosphate results in the degradation of the end of the S-RNA 

 and in an increased incorporation of cytidylic acid, it is reasonable to expect 

 that new sites in the S-RNA may be exposed. If these new sites accept 

 cytidylic acid, then incubation of the pyrophosphorylyzed S-RNA with 

 p2p]-CTP (32P-P-P) followed by alkaline hydrolysis of the CM^sp- 

 labelled S-RNA should result in the identification of the site of attachment 

 of the cytidylic acid. We have performed this experiment under conditions 

 of low and maximal pyrophosphorolysis of S-RNA, and compared the 

 results with those obtained with non-pyrophosphorylyzed S-RNA. Pyro- 

 phosphorolysis results in the unveiling of adenylic acid as the new site of 

 attachment of p-P]-CMP. The distribution pattern of the ^^P obtained 

 with maximally pyrophosphorylyzed S-RNA (Table 5) shows the ^^P to 

 be equivalently distributed between 2' (3')-cytidylic acid and 2' (3')- 

 adenylic acid. These results make the following interpretation plausible: 



P-P + AMP— CMP— CMP— AMP— S-RNA -> 



AMP— S-RNA + ATP + 2CTP 



32P.CTP + AMP— S-RNA -> 



32P-CMP—32p.CMP— AMP— S-RNA + P-P 



In other words, pyrophosphorolysis of S-RNA has exposed a terminal 

 adenylic acid which now accepts two cytidylic acid residues. This S-RNA 

 upon alkaline hydrolysis would yield equivalent amounts of P^pj.cytidylic 

 acid and adenylic acid. Ti is would therefore indicate adenylic acid to be 

 the fourth ribonucleotide in the terminal sequence. Although this inter- 

 pretation is in keeping with the observed facts it can in no way be con- 

 sidered to be the only possible interpretation. We therefore wish to suggest 

 this as a temporary explanation, reserving final judgment until a more 

 homogeneous preparation of S-RNA is available. 



This interpretation of these results raises the question as to the source 

 of the guanosine triphosphate and of the uridine triphosphate found in the 

 acid-soluble fraction after pyrophosphorolysis of the S-RNA. We believe 

 that since guanylic acid and uridylic acid exist as terminal ribonucleotides 

 in our S-RNA preparations (Fig. 4), these may well be pyrophosphorylyzed 

 in a manner similar to that in which the terminal adenylic acid and cytidylic 

 acid are pyrophosphorylyzed. This is supported by the fact that pyro- 

 phosphorylyzed S-RNA as compared to normal S-RNA, also shows an 

 enhanced incorporation of uridylic acid similar to that of cytidylic acid. 



