386 MAHLON B. HOAGLAND 



at pH's below neutrality even at a high temperature, and in alkali it 

 hydrolyzed at a rate considerably slower than amino acyl phosphoanhy- 

 drides. It was also much less reactive with hydroxylamine and ammonia. 

 The periodate inactivation studies mentioned above were also strongly 

 indicative of an ester bond between amino acid carboxyl and ribose-2'- 

 or 3'-hydroxyl group. As was mentioned above, the nature of the linkage 

 has now been established in both mammalian 178 and bacterial 131 systems 

 by direct removal by ribonuclease of the terminal adenosine-amino acid 

 ester. Thus, the configuration of the terminal grouping illustrated in Eq. 

 (5) appears to be correct. 



This particular type of linkage is of special interest because it is in 

 equilibrium with the AMP pyrophosphoryl bond of ATP via the steps 

 in Eqs. (4) and (5). For example, Schweet 181 has obtained an equilibrium 

 constant of 0.4 for the reaction 



ATP -(- RNA + threonine ^ threonine - RNA + AMP + PP 



Similarly Berg's group found a value of 0.32 for valine RNA formation. 177 

 A simple ester bond would certainly not be expected to have such a high 

 energy content and we must therefore assume that the presence of the 

 adjacent hydroxyl group on the ribose confers special reactivity properties 

 to the linkage. 



Naturally occurring sRNA derived from mammalian pancreas 178 and 

 liver 14 ' 182 and E. coli (cf. Chapter 38) has been found to have amino acids 

 bound to it. Analysis indicates that nearly all of the amino acids are 

 represented, 182 and that the sRNA is actually saturated with amino acids. 14 

 Preliminary studies indicate that this is also true for yeast transfer RNA. 126 

 The E. coli transfer RNA preparation of Berg's group, on the other hand, 

 is reported to be free of amino acids. 131 This may be due to loss during 

 the preparative procedure. The "pressure head" of ATP in cells would 

 serve to saturate the transfer RNA pool with amino acids as soon as they 

 entered the cell. Thus, transfer RNA is an activated amino acid pool of 

 not inconsiderable dimensions. 



d. Evidence for a Direct Role of Transfer RNA in Protein Synthesis 



The evidence that a particular cellular RNA fraction binds amino acids 

 specifically suggests the likelihood that it may be involved in the mecha- 

 nism by which these amino acids ultimately find their way into protein. 

 The existence of these RNA-amino acid reactions in a tissue component 

 required for incorporation of amino acids into protein further supports 



181 R. S. Schweet, unpublished data, (1959). 



182 G. Acs, G. Hartmann, H. G. Boman, and F. Lipmann, Federation Proc. 18, 700 

 (1959). 



