388 MAHLON B. HOAGLAND 



siderably more detailed studies on the in vivo interrelationship of these 

 fractions have been carried out by Lacks and Gros. 124 These investigators 

 have confirmed the mammalian results in an E. coli system and have 

 elegantly established two further points: (1) the rate of attachment of 

 amino acids to transfer RNA is a function of the rate of protein synthesis. 

 Inhibition of protein synthesis slows the rate of equilibration of a C 14 - 

 amino acid with the transfer RNA pool but does not alter the final equi- 

 librium level. Amino acids of which the cells have been deprived equilibrate 

 much more rapidly with the pool. These findings suggest that the availa- 

 bility of sites on transfer RNA is a direct function of the rate at which 

 the sites are made available by subsequent steps in protein synthesis. 

 (2) Removal of the inhibition of protein synthesis results in a transfer 

 of the labeled amino acid from the RNA pool to protein. These matters 

 are considered more fully in Chapter 38. 



Brachet 183 has recently contributed to the growing body of evidence sup- 

 porting the role of transfer RNA in protein synthesis. Extending his earlier 

 studies on the ribonuclease inhibition of protein synthesis in onion root tips, 

 he has shown that in the inhibited system, it is the sRNA that is depleted, 

 while ribosomal RNA is relatively stable. It would bo safe to conclude that 

 the in vivo kinetic behavior of transfer RNA suggests that it could be an 

 intermediate in protein synthesis. 



When we turn to the cell-free systems available to us we find we are 

 only on the verge of discovery, but what has come to light, thus far, 

 further supports the role of transfer RNA amino acid as an intermediate. 



It was found early that transfer RNA labeled with amino acids in the 

 whole pH 5 fraction would transfer the amino acids to microsome protein 

 in the presence of ATP, GTP, and a nucleoside triphosphate generating 

 system 117 (see Fig. 5). Furthermore, transfer RNA, properly labeled with 

 C 14 -amino acids by activating enzymes and ATP, may be isolated by the 

 phenol method and alcohol precipitation as a fairly pure group of com- 

 pounds, free of protein and adsorbed amino acids. Incubation of this 

 material with microsomes, Mg ++ , ATP, an ATP generating system, GTP, 

 and an enzymic component of the soluble cell fraction, [the S 4 fraction 

 described in Section II, 2, a, (1)], resulted in a rapid and irreversible 

 transfer of the amino acid to ribosomal protein 117 (see Fig. 6). The omission 

 of any of these components gave low activity. The amino acid in this 

 protein was not found in terminal positions but within the peptide chain. 96 

 (This is in contrast to the more recent results of Webster 166 who finds, 

 starting with glutamyl-C 14 RNA and methionyl-C 14 RNA, that the amino 

 acids are reversibly incorporated into pea seedling ribosomal protein and are 

 in Af -terminal positions.) It could be demonstrated that when transfer 



183 J. Brachet, Biochim. et Biophys. Acta 35, 580 (1959). 



