INTERMEDIATE REACTIONS IN PROTEIN SYNTHESIS 1 63 



Although several of these have been isolated, the possibility of a separate activat- 

 ing enzyme for each amino acid remains to be confirmed. This reaction is not 

 sensitive to ribonuclease, as is the over-all incorporation process. The ubiquity of 

 the reaction makes it an attractive possibility that this activation of the carboxyl 

 group of amino acids is an initial step in protein synthesis. 



Another reaction possibly involved in the over-all incorporation of amino acid 

 into protein was found quite unexpectedly. Because of the close association ot 

 RNA with the initial labeling of protein, wc tried to correlate RNA and protein 

 synthesis in this enzyme system. Labeled 8-C'^-ATP and labeled amino acid were 

 incorporated separately in the cell-free liver system; then both the RNA and 

 protein were isolated to see how much of the ATP label had appeared in the 

 RNA, and how much amino acid was found in the protein. Surprisingly, the 

 RNA incorporated not only the labeled nucleotide, but also leucine-C*. 



This binding of amino acid to RNA is being investigated by a number of 

 workers (23, 22, 41, 4, 34, 39, 24). Further studies have shown that it is the RNA 

 present in the soluble fraction of the cell (S-RNA) to which the amino acid 

 becomes bound. 



S-RNA, isolated by phenol extraction, retains its capacity to bind amino acid 

 when added to the incubation system containing the supernatant fraction (22). 

 Neither microsomal nor nuclear RNA is able to bind the amino acid to any 

 great extent when added. 



Studies of the binding of individual, and mixtures of, amino acids to the S-RNA 

 indicated that there is no competition between the individual amino acids for 

 sites on the S-RNA, since the binding of each amino acid is unaffected by the 

 presence of other amino acids. 



The soluble RNA (S-RNA) is conveniently concentrated in the pH 5 fraction 

 mentioned above. The necessary enzymes for these incorporation reactions are 

 also present in the pH 5 fraction, although not exclusively so. 



A very specific requirement for ATP and cytosine triphosphate (CTP) has been 

 demonstrated for the binding of the amino acid to this unique type of RNA 

 (S-RNA). This requirement could not be demonstrated in the untreated pH 5 

 fraction, but following incubation of the pH 5 fraction for 20 minutes at 37°C 

 in the absence of added nucleoside triphosphates and ainino acids it became pos- 

 sible to demonstrate a dependence of the amino acid binding to S-RNA in the 

 presence of CTP and ATP ( 15, 16). With untreated pH 5 fraction the binding of 

 valine to S-RNA is not dependent upon the presence of CTP. However, when 

 the pH 5 fraction is preincubated as described, the amino acid is not bound to 

 S-RNA unless both CTP and ATP are present. Neither GTP nor uridine triphos- 

 phate (UTP) can replace CTP in this reaction and an absolute requirement for 

 ATP is indicated (22). The CTP requirement has been observed for all 14 

 separate amino acids which have been tested. 



