164 SUBCELLULAR PARTICLES 



The role played by CTP in binding amino acid to S-RNA has been elucidated 

 by studies with labeled nucleotides. Our earlier attempt to correlate RNA syn- 

 thesis with protein synthesis indicated that the same S-RNA which could bind 

 amino acids also was able to incorporate C^ ^-labeled nucleotides. The nucleoside 

 triphosphates were the precursors of the nucleotide incorporated into the S-RNA 

 (45, 17, 6, 13, 18). ATP, CTP and to a lesser extent UTP were all precursors 

 for the incorporation of nucleotides into S-RNA. GTP, however, was not 

 utilized. The reaction mechanism appears to be as indicated: 



ATP + RNA^AMP-RNA + PP 



This incorporation is inhibited by pyrophosphate, and the reversibility has been 

 demonstrated by an S-RNA dependent exchange of P'^- into added CTP (17). 



As with the binding of amino acids, the incorporation of the nucleotide was 

 specific for S-RNA. Added nuclear and microsomal RNA were inert. This is 

 shown in figure 2. A CTP requirement for the incorporation of the adenine 

 nucleotide was demonstrated with the preincubated pH 5 fraction described 

 previously (17). 



Alkaline hydrolysis of the S-RNA isolated after incubation with C^^-ATP and 

 C^^-CTP was used to indicate the positions of the nucleotide in the S-RNA chain. 

 Mild alkaline hydrolysis of RNA degrades the molecule uniformly yielding 

 3'(or 2') mononucleotides from the internal nucleotides of RNA, and nucleosides 

 are liberated from one end of the chain. S-RNA isolated from the preincubated 

 pH 5 fraction after incubation with C^^-ATP and/or C^'-CTP revealed a se- 

 quential incorporation of nucleotide into the S-RNA (17). As figure 3 indicates, 

 the cytosine nucleotides and a terminal adenine nucleotide are added in sequence 

 to 'apo-RNA.' The 'apo-RNA' is that isolated from the preincubated pH 5 frac- 

 tion while the RNA isolated from the untreated supernatant fraction contains 

 the terminal nucleotides and amino acids. 



The CTP dependence of the binding of amino acid to the S-RNA indicates 

 that the terminal groups on the end of the S-RNA chain play a role in the 

 attachment of the amino acid to this type of RNA (16). Figure 4 indicates the 

 available positions on this terminal end of the RNA. The existence of an ATP 

 containing an amino acid on the 2' (or 3') hydroxyl of the ribose described by 

 Weiss, Acs and Lipmann (40) provides some evidence that the same positions 

 on the terminal ribose unit on S-RNA would be feasible in this type of molecule.* 



Our evidence suggests that under certain experimental circumstances there are 



* Note added in proof: Since the presentation of this paper more direct evidence has indicated 

 that the amino aciil is linked to the 2' (or 3') hydroxyl group of the terminal adenine 

 nucleotide of S-RNA. (Zachau, H. G., G. Acs and F. Lipmann. Proc. Nal. Acad. Sci. 44: 885, 

 1958; Hecht, L. I., M. L. Stephenson and P. C. Zamecnik. Proc. Nat. Acad. Sci. In press. 

 Preiss, J., P. Berg, E. J. Oi engand, F. H. Bergmann and M. Dieckmann. Proc. Nat. Acad. 

 Sci. In press. 



