358 RICHAHI) SCIIWKKT AM) JOIIX BISHOP 



C SPI'^riFICITV OF AMINO ACVL-RNA FORMATION 



Thr sp(M'ifi('ity of aiuino acid-activating enzyincs for individual 

 aniiiio acids was demonstrated soon after tiie discovery of these enzymes. 

 A number of enzymes which activate only a single amino acid have been 

 isolated (reviewed by Raacke, 1961; Berg, 1961). Although analogs of 

 the amino acid may be activated by a i)articular enzyme, in most cases 

 only a single naturally occurring amino acid is activated by a particular 

 enzyme. An excejition is the isoleucine enzyme studied by Bergmann 

 ft (d. (19G1) which activated valine also when measured by PP-ATP 

 exchange. However, even this enzyme formed only isoleucyl-RNA, a 

 more significant assay for the physiological functioning of these enzymes 

 (Berg et al., 1961). These results thus indicate a two-stage specificity 

 in the action of activating enzymes. First, a specificity for the formation 

 of the enzyme-amino acyl-adenylate complex; and second, a specificity 

 of the complex in its reaction with T-RNA. This second specificity 

 extends to the recognition of T-RNA molecules specific for individual 

 amino acids. 



Despite the uniformities in structure at the ends of T-RNA, evidence 

 has accumulated that fractions specific for the attachment of individual 

 amino acids are present. Evidence for T-RNA fractions specific for 

 individual amino acids based on fractionation was first noted by Schweet 

 et al. (1958a). Fractionation procedures using untreated T-RNA have 

 resulted in partial separations of fractions specific for various amino 

 acids. Chromatography on an anionic starch resin (Smith et al., 1959) , 

 countercurrent distribution (Holley and Merrill, 1959), clectroi:)horesis 

 (Lipmann et al., 1959), and chromatography on calcium phosphate 

 columns of various types (Hartmann and Coy, 1961 ; Cantoni, 1962) 

 have been used. None of these methods has so far resulted in isolation 

 of a fraction specific for a single amino acid.'' Promising results have 

 also been obtained by utilizing the change in jn'operties of a particular 

 RNA by virtue of having its amino acid attached. Tyrosyl- and histidyl- 

 RNA chains have been ]:)artially purified by complexing to i)olydiazo- 

 styrene columns (Brown et al., 1959). What appears to be a nearly pure 

 RNA fraction specific for valine has been reported by Stephenson and 

 Zamecnik (1961). Yeast soluble RNA containing only attached valine 

 was prepared. Aftci- oxidation by pcriodate of the RNA (the valyl-RNA 

 was protected), and attachment of a bulky dye molecule, fractionation 

 on DEAE-Sephadex yielded a fraction which in one experiment was 



'Holley (private eommunicalion) has rri)orte(l tlie isolation of amino arid- 

 specific T-RNA fractions using countercurrent distribution. 



