VIII. PROTEIN SYNTHESIS AND GENE ACTION 363 



ribosonie followed by the appearance of the completccl protein in the 

 nonsedimentable fraction. 



A. TRANSFER OF AMINO ACIDS FROM AMINO ACYL-RNA TO RIBOSOMES 



1. Amino Acyl-RN A as an Intermediate in Protein Synthesis 



The transfer of amino acids from amino acyl-RNA compounds to 

 ribosomal protein indicated that these compounds were intermediates in 

 protein synthesis (Hoagland et al., 1958). Evidence for a soluble inter- 

 mediate in protein synthesis had been reported earlier by Hultin (1956). 

 The isolation of labeled hemoglobin (Bishop et al., 1961 ; von Ehrenstein 

 and Lipmann, 1961) and albumin (Hirokawa et al., 1961; Campbell, 

 1961, 1962), starting with C^^-amino acyl-RNA, provided strong evidence 

 that these compounds could serve as intermediates for the synthesis of 

 defined proteins. 



Kinetic studies w'ith intact cells supports the role of amino acyl-RNA 

 compounds as major intermediates in protein synthesis. The work of 

 Hoagland et al. (1958) showed that, in ascites cells at depressed tem- 

 peratures, labeled amino acids were initially linked to soluble RNA more 

 rapidly than they were found in peptide linkage either in the microsome 

 fraction or in soluble protein. Lacks and Gros (1959), using intact 

 E. coli cells, also showed that the soluble RNA fraction was most 

 rapidly labeled. In addition, the experiments were compatible with the 

 idea that the label was passed on into peptide linkage when the labeled 

 amino acid (S'^°-methionine) was diluted with a fivefold excess of non- 

 radioactive methionine. When chloramphenicol was added to the incuba- 

 tion, greatly reducing the rate of protein synthesis, the rate of equilibra- 

 tion of the RNA-bound amino acid with the free amino acid pool was 

 also reduced, although the same saturation point was ultimately reached, 

 suggesting that transfer of the RNA-bound amino acid to protein was 

 the normal pathway which released free RNA for further formation of 

 RNA-bound amino acid. Lacks and Gros (1959) feel that amino acyl- 

 RNA is an intermediate in protein synthesis, but that amino acids may 

 also extend protein by a second pathw^ay. The hypothesis is based on the 

 observation that the initial rate of labeling of the RNA-bound amino 

 acid was one-third as fast as the initial rate of entiy of the label into 

 ''protein." Roberts (1959) has indicated that the turnover rate of amino 

 acyl-RNA was too small for these compounds to act as the only inter- 

 mediate in protein synthesis. These results are consistent with amino 

 acyl-RNA compounds acting as intermediates in protein synthesis in 

 the intact cell, but suggest the existence of other pathways which could 

 be of considerable importance. On the other hand, no other pathway for 



