356 RICHARD SCHWEET AND JOHN BISHOP 



Reaction 1 can be measured by the amino aci(i-(le])en(lent incoij)ora- 

 tiun of P^--pyrophosphate into ATP and by the formation of amino acyl 

 liych'oxamates wlien hirge amounts of hydroxyhunine are present. The 

 evidence for the formation of the enzyme-bound AMP-amino acid, in 

 which tiie phosphate is linked in anhydride bond with the amino acid 

 carboxyl, is quite conchisive. This intermediate has been isohited using 

 substrate amounts of enzyme and its synthesis shown by 0^^ transfer 

 experiments. 



Reaction 2 has been demonstrated directly starting with tlie enzyme- 

 AMP-amino acid complex, but is usually measured using the over-all 

 reaction 3. The incorporation of a C^^-amino acid into T-RNA (transfer 

 RNA, see below) is measured. This reaction is the physiological one 

 catalyzed by these enzymes and they have recently properly been named 

 amino acyl-RNA synthetases (Berg, 1961). The evidence that reactions 

 1 and 2 are catalyzed by the same enzyme is based largely on the failure 

 to separate these activities on j^urification of the enzyme (Berg and 

 Ofengand, 1958). Later studies are in agreement with these results, in- 

 cluding those with an alanine enzyme where the activity for PP :?± ATP 

 and AMP ^ ATP exchange were purified together (Holley and Gold- 

 stein, 1959). The reversibility of reaction 3 has been demonstrated and 

 the equilibrium constant determined from both directions. The values 

 obtained for various amino acids indicate the high-energy nature of the 

 amino acyl-RNA bond (Glassman et al., 1958; Lipmann et al., 1959; 

 Preiss et al., 1959; Leahy et al., 1960). The specificity of amino acyl- 

 RNA synthetases is discussed below. The review by Stulberg and 

 Novelli (1962) contains further details of these reactions. 



B. TRANSFER RNA 



The RNA which is active for formation of amino acyl-RNA com- 

 pounds has been called transfer RNA (T-RNA) based on its function in 

 protein synthesis (Allen et al, 1960) or soluble RNA (S-RNA) based 

 on its location in the cell (Hoagland, 1960). The term T-RNA has been 

 used recently to distinguish this RNA from another type of RNA, also 

 found in the soluble cytoplasm under some conditions, that is, messenger 

 RNA. T-RNA is a specific type of RNA with a molecular weight of 

 about 25,000. However, T-RNA does not appear to be either a precursor 

 or degradation product of larger molecular weight RNA. This conclusion 

 is based on the unique structure of T-RNA. In particular, T-RNA has 

 known end groups as shown: 



pGp RNA pCpCpA 



