l6y SUBCELLULAR PARTICLES 



Table 2. Enhancement by ctp of incorporation of c"-valine into liver 



PROTEIN and RNA 



rPM PtR MG 



Protein RNA 



ATP 8.6 983 



ATP, GTP 20.0 992 



ATP, GTP, CTP 63.3 1360 



The incubation system contained microsomes centrifuged from a dilute 15,000 g liver super- 

 natant fraction, soluble enzymes treated with Dowex-i-Cl to remove nucleotides, preincubated 

 pH 5 fraction, o.imM valine-i-C* containing 300,000 cpm/ml, 0.005 m ATP and a nucleoside 

 triphosphate generating system. The indicated nucleotides were added at final concentration of 

 0.1 yUM. The total volume was i ml. Incubation was for 15 min. at 37° C. 



SUMMARY 



The relationship of the isolated steps described herein is illustrated in table 3. 



Table 3. Postulated steps in incorporation of amino acids into protein 



1. Activation of amino acids: 



AMINO ACID + ATP + ENZYME ^ [ AA~AMP] - ENZYME + PP 



2. Addition of nucleotide end groups to S-RN.-\: 



RNA \- ATP + CTP ^ RNA - pCpCpA + PP 



J. Binding of amino acids to this S-RNA (enzymes may be identical with activating enzymes) : 

 AA + ATP + RNA - pCpCpA ;^ RNA - pCpCpA - AA + PP 



4. Transfer of amino acids to ribonucleoprotein particles of microsomes, with binding to ribo- 



nucleoprotein RNA, followed by polymerization of amino acids: 

 RNA - pCpCpA - AA + ATP + GTP + RNP - particles + soluble fraction -» peptide chain 



5. Release of bound peptide chain and completion of protein by cross linking and secondary 



bonding 



It should be stressed that much of the evidence is indirect, and some of the reac- 

 tions described might be quite remote from the direct path of protein synthesis. 



The first step, an activation of the amino acids, does not involve RNA. The 

 reaction specifically requires ATP and cannot be replaced by CTP, UTP, or GTP. 

 There are apparently many specific enzymes. 



In another series of reactions, a unique type of soluble RNA (S-RNA) receives 

 specific nucleotide end groupings. This reaction is independent of amino acids. 



A third reaction binds amino acid to the S-RNA, which must contain the 

 specific nucleotide end groups. There is no competition among the amino acids, 

 indicating the presence of a specific binding site or a specific S-RNA molecule 

 for each natural amino acid. This reaction may be carried out by the activating 

 enzymes. 



Evidence has been presented for a transfer of the S-RNA bound amino acid 

 to both microsomal RNA and to microsomal protein. At this point we are on 

 less stable ground. Whether the dependence upon GTP applies only to the bind- 



