37. NUCLEIC ACID AND PROTEIN SYNTHESIS 385 



radative studies to reveal the structural characteristics of the RNA mole- 

 cules which underlie their specificity. 



(3) The Terminal Nucleotide Grouping as a Requirement for Amino Acid 

 Attachment. Because of the reversibility of attachment of the terminal 

 nucleotide groups to transfer RNA it became possible to "strip" the RNA 

 of these nucleotides by incubating it with the terminal nucleotide system 

 and PP. This "stripped" RNA could then be tested for its ability to accept 

 amino acids in the presence of ATP, CTP, or both. 



Careful exploitation of this technique by Hecht et a£. 155, 179 has revealed 

 that both ATP and CTP are necessary to relabel such "stripped" RNA 

 with C 14 -amino acids. That the added requirement for CTP is due to 

 the reattachment of this nucleotide's CMP moiety to the end of the RNA 

 was shown by preincubating the RNA with CTP and the enzyme system 

 which attaches CMP to the end of the RNA. This RNA, when reisolated, 

 was then able to accept amino acids, like the naturally occurring material, 

 in the presence of ATP alone. The requirement for both CTP and ATP 

 in the labeling of "stripped" RNA was shown to be common to all amino 

 acids. It therefore seems highly probable that the same type of terminal 

 configuration of nucleotides is required for attachment of all amino acids 

 to their specific RNA molecules. 



It should perhaps be stated explicitly that the terminal AMP grouping 

 of the RNA to which the amino acid becomes attached is not derived 

 from the AMP moiety of the amino acyl adenylate which furnishes the 

 amino acid. This was considered a possibility at one time but is ruled 

 out by the facts that (1) terminal AMP attachment to RNA is clearly 

 catalyzed by enzymes other than those which activate amino acids 123 ; 

 (2) in crude systems containing both activities terminal AMP attachment 

 is not dependent on amino acids 155 ; (3) the optimal ATP concentration 

 for AMP attachment is lower, by a factor of 100, than the optimal ATP 

 concentration for amino acid attachment. 155 



(The anomalous observation of Lipmann, 180 that in the presence of 

 tryptophane,-activating enzyme and ATP, tryptophan-C 14 could esterify 

 the ribose-2' (3') -hydroxy 1 of ATP suggested a mechanism by which both 

 amino acid and AMP could be attached to transfer RNA in a single step. 

 This seems improbable in the light of the above evidence and the reaction 

 probably represents an unphysiological phenomenon resulting from the 

 excess of ATP present and the absence of a natural acceptor for the amino 

 acid.) 



(4) The Site of Amino Acid Attachment. It was apparent, early, that 

 the chemical behavior of the RNA-amino acid link seemed to preclude 

 its being a phosphoanhydride type of bond. The bond was quite stable 



180 S. B.Weiss, H.G.Zachau, and F. Lipmann, Arch. Biochem. Biophys. 83, 101 (1959). 



