102 THE BIOSYNTHESIS OF PROTEINS 



particles there was a big gap. Since the amino acid adenylates are strongly- 

 bound to the activation enzymes, the problem was to find out how activated 

 amino acids were transferred to the microsomal particles. 



Hultin (1956) obtained evidence for the accumulation in homogenates 

 of some form of activated amino acids which can serve as precursors of 

 proteins in the absence of ATP. The addition of a one thousand-fold excess 

 of non-labelled leucine to a homogenate in which radioactive leucine is 

 being incorporated causes a sudden and radical drop of specific radio- 

 activity of the free leucine, but incorporation is not stopped immediately: 

 it goes on undisturbed for a few minutes, before being completely inhibited. 

 This indicates that some intermediate between free amino acids and 

 precipitable protein piles up in the homogenate. The intermediate is in the 

 soluble fraction, and it is in a form which does not equilibrate rapidly with 

 free amino acids as the amino acid adenylates would do (Hultin and 

 Beskow, 1956). 



Holley (1957) obtained indications on the probable polyribonucleotidic 

 nature of the acceptor by observing that ribonuclease suppresses a AMP- 

 ATP exchange which is catalysed by the pH 5 fraction and which depends 

 on alanine. Hoagland et ah (1957) found that the 'pH 5 enzymes' prepara- 

 tion from rat liver contains about 5 per cent ribonucleic acid, and that 

 when this preparation is incubated with ATP and labelled leucine, the 

 RNA subsequently isolated from this fraction is labelled. Leucine is thus 

 bound to the RNA of the pH 5 fraction. The bond is relatively stable in 

 acid medium, and alkali labile. Bound leucine does not exchange with non- 

 labelled free leucine. When the leucine-labelled RNA is incubated with 

 concentrated hydroxylamine, leucylhydroxamic acid is formed, indicating 

 that leucine is bound to RNA through the carboxyl in such a way that this 

 group is moderately reactive. When leucine-labelled RNA, isolated by the 

 phenol method (Kirby, 1956) is added to a microsomal suspension, leucine 

 leaves the RNA and it is transferred to microsomal protein material, pro- 

 vided guanosinetriphosphate is present (Hoagland et al., 1957, 1958; 

 Zamecnik et al., 1958). 



Thus some ribonucleic acid which is contained in the soluble fraction, 

 and in the pH 5 precipitate obtained therefrom, can act as a transitory 

 carrier of activated amino acids in between the activation step catalysed by 

 the activation enzymes, and the microsomal particles where polypeptides 

 first appear. 



2. Transfer RNA 



The discovery of amino acid binding by soluble RNA from rat liver 

 was soon confirmed, and RNAs with similar acceptor properties were 

 found in other animal tissues, e.g. pancreas (Weiss et al, 1958), mammary 

 gland (Fraser and Gutfreund, 1958), in a protozoan (Mager and Lipmann, 



