VIII. PROTEIN SYNTHESIS AND GENE ACTION 379 



used, since ATP is not required, a better assessment of the energy re- 

 quirements for the formation of soluble protein may be made. Bishop 

 et al. (1961) found that little soluble protein was formed in the transfer 

 system, with GTP as the only nucleoside trijihosphate. Howevei", this 

 was probably due to the small amount of C^^-amino acyl-RNA used in 

 these experiments. Formation of soluble protein using a transfer system 

 was shown by von Ehrenstein and Lipmann (1961). However, since 

 GTP plus a generating system was used, it is likely that ATP was 

 formed. In any event, since chains were being formed, the "release" 

 mechanism per se was not studied in either of these cases. Even the 

 formation of soluble protein from labeled ribosomes with GTP and 

 enzyme in the absence of amino acyl-RNA (Table III) does not repre- 

 sent release alone, since it is possible that chain growth from intraribo- 

 somal intermediates occurs here. Thus, other than the evidence which 

 suggests that some mechanism is involved in liberating completed pro- 

 teins from the ribosome, practically nothing is known. 



Studies of the mechanism of inhibition of protein synthesis by the 

 antibiotic puromycin have been of interest in this connection. Inhibition 

 of protein synthesis puromycin was first reported by Yarmolinsky and 

 de la Haba (1959). Inhibition of transfer from amino acyl-RNA to 

 ribosomes was considered to be the site of action of puromycin. This 

 seemed likely since puromycin has a structure analogous to the amino 

 acyl-adenosine end of transfer RNA. A direct effect of puromycin on 

 the ribosome resulting in the release of soluble protein has been reported 

 for reticulocyte ribosomes (]Morris and Schweet, 1961) and liver ribo- 

 somes (Hultin, 1961). It was later shown that release of soluble protein 

 by puromycin was maximal in the absence of added soluble enzyme and 

 energy (jVIorris et al., 1962). Release of soluble protein occurred at 4°C, 

 although more slowly than at 37° C. Ribosome breakdown could not be 

 detected under these conditions. The protein material released by puro- 

 mycin in the absence of added enzjane was shown to be incomplete globin 

 chains synthesized from the N-terminal end, confirming earlier conclu- 

 sions (Section III,B,2). 



In the presence of soluble enzymes and puromycin, in addition, free 

 amino acids and small peptides are found in the TCA-soluble fraction. 

 These have not been well characterized, but it has been suggested that 

 they may represent earlier, intraribosomal intermediates in globin syn- 

 thesis (Morris et al., 1962). The inhibition of protein synthesis by 

 puromycin is considered to result from the displacement of incomplete 

 globin chains and the postulated earlier intermediates from the ribosome. 

 In the ]:)resence of puromycin, further formation of TCA-precipitable 

 peptides would be blocked by a continuation of the same process. This 



