372 MAHLON B. HOAGLAND 



acids in a highly specific manner. 116, 117 Such reactions were independently 

 discovered by Ogata et al. in Japan. 118 ■ 119 



That a stable intermediate might be formed on the path between free 

 amino acid and protein was first suggested by Hultin and Beskow. 120 These 

 workers found that incubation of a crude supernatant fraction of rat liver 

 with C 14 -amino acid and ATP led to a conversion of the C 14 -amino acid to a 

 state in which it no longer equilibrated with free C 12 -amino acid. Holley 121 

 furthermore, had pointed out that a similar crude fraction catalyzed a 

 ribonuclease-sensitive, alanine-dependent incorporation of adenosine mono- 

 phosphate-C 14 (AMP-C 14 ) into ATP, suggesting the formation of an RNA- 

 amino acid intermediate. 



These matters have now been examined by many investigators and the 

 general picture has emerged that the amino acid activating enzymes not 

 only catalyze the activation of amino acids by the mechanism postulated, 

 but also attach the amino acid so activated through an ester linkage to the 

 terminal nucleotide of specific soluble RNA molecules. These molecules, 

 once charged with amino acid, have been found to be able to transfer the 

 amino acid to the protein of the ribonucleoprotein particles under appro- 

 priate conditions. 116 ' m Such soluble RNA-amino acid compounds, which 

 thus satisfy several criteria for intermediates in the incorporation process, 

 have become another strong link in the chain of evidence implicating RNA 

 in protein synthesis. This interesting species of RNA and the reactions in 

 which it participates must now be discussed. 



a. Physicochemical Aspects of Transfer RNA 



(1) Occurrence and Methods of Preparation. "Soluble RNA" (sRNA) or 

 "cell sap RNA" are terms used to designate that fraction of total cellular 

 RNA, usually 10 to 20%, isolatable by methods which depend on the rela- 

 tively low molecular weight of the material. Usually this means the frac- 

 tion which remains unsedimented after cent rif ligation of a tissue homoge- 

 nate at 100,000 g for 1-2 hours. It would also, most likely, include material 

 relatively more soluble in salt than the bulk of the cytoplasmic RNA. 

 Such preparations contain more RNA than that responsible for amino acid 

 binding, due to the presence of degraded microsomal material and perhaps 

 other RNA of unknown significance. It has been suggested by Schweet 



116 M. B. Hoagland, P. C. Zamecnik, and M. L. Stephenson, Biochim. et Biophys. 

 Acta 24, 215 (1957). 



117 M. B. Hoagland, M. L. Stephenson, J. F. Scott, L. I. Hecht, and P. C. Zamecnik, 

 ./. Biol. Chem. 231, 241 (1958). 



118 K. Ogata and H. Nohara, Biochim. el Biophys. Acta 25, 660 (1957). 



119 K. Ogata, H. Nohara, and T. Morita, Biochim. el Biophys. Acta 26, 657 (1957). 



120 T. Hultin and G. Beskow, Exptl. Cell Research 11, 664 (1956). 



121 R. W. Holley, J. Am. Chem. Soc. 79, 658 (1957). 



