384 MAHLON B. HOAGLAND 



more sites than one available on the RNA for methionine attachment, 

 the yeast enzyme may "recognize" only one site while the E. coli enzyme 

 can recognize this and another. 



We may tentatively conclude that the activating enzymes catalyze a 

 two-step reaction when transfer RNA is present — formation of an acyl 

 adenylate and attachment of the acyl moiety to transfer RNA. 



(2) Individual RNA's as Acceptors of Individual Amino Acids. Early 

 observations of amino acid-sRNA interaction indicated that amino acids 

 were additively and noncompetitively attached to sRNA. This clearly 

 established that separate sites were available for each amino acid but 

 did not tell us whether separate RNA's were involved. Zachau et al. m 

 then demonstrated directly that the amino acids were esterified on the 

 terminal adenosine of the RNA. This was done by treatment of leucine 

 labeled sRNA with pancreatic ribonuclease, which resulted in a quanti- 

 tative yield of leucyladenosine. It had been shown by Hecht et a/. 155 ' 179 

 that the terminal configuration of nucleotides pCpCpA was necessary for 

 amino acid attachment (see below). These workers and Preiss et al. m 

 also showed that periodate treatment of the sRNA resulted in loss of 

 ability to accept amino acids and that amino acids protected against the 

 action of periodate. Periodate would be expected to oxidize any free 2'-, 

 3'-hydroxyl groups. Preiss et al. m carried the periodate experiment a step 

 further by showing that amino acids could protect only their own sites 

 of attachment to sRNA from periodate oxidation. 



Hecht et al., furthermore, demonstrated that the ordinarily free reversi- 

 bility of the reaction by which the terminal pCpCpA grouping was at- 

 tached to the RNA was markedly inhibited by amino acids. 155 This sug- 

 gested that the amino acids might, in fact, actually be attached to this 

 terminal grouping, thereby preventing the nucleotide end group from 

 reacting with PP. Thus there was strong indication that the terminal 2'-, 

 3'-hydroxyl groups of the polynucleotide chains were involved in the link 

 with amino acids. 



These experiments supplemented and supported the observations of 

 Schweet's group (cf. Symposium 13141 ) that crude fractionation of sRNA 

 resulted in enrichment of fractions for acceptance of one amino acid over 

 another. This series of observations has, thus, strongly stimulated the 

 search for better means of fractionating sRNA, alluded to earlier, which 

 is likely, ultimately, to result in the isolation of several homogeneous trans- 

 fer RNA species each capable of reacting with only one amino acid. When 

 this has been accomplished it should then be possible to undertake deg- 



178 H. G. Zachau, G. Acs, and F. Lipmann, Proc. Natl. Acad. Set. U. S. 44, 885 (1958). 



179 L. I. Hecht, M. L. Stephenson, and P. C. Zamecnik, Biochim. et Biophys. Acta 29, 

 •160 (1958). 



