The Effect of Bisulfite - induced C + U Transitions on Aminoacylation of Escherichia coli Glycine tRNA

The effect of bisulfite-induced C + U transitions on the aminoacylation of highly purified Escherichia coli tRNAt,’& has been studied. On treatment with 3.2 M NaHS03 at 25 “C and pH 5.8, C + U transitions occurred at 4 of the 21 cytosine residues during the first 16 h. After about 12 h, 2 additional unidentified residues begin to react. From these data and theoretical consideration, we conclude: 1) E. coli tRNA:’&, exists in the native conformation under the conditions of the reaction; 2) its ordered structure is similar to yeast RNAPhe; 3) its anticodon loop is flexible in solution. During the reaction, loss of glycine acceptor activity followed first order kinetics with a t lIz = 1.8 h. After modification for 1.25 h and aminoacylation (61% glycine acceptor activity remaining), the aminoacylated fraction was isolated. The fractional change at each of the reactive residues in the unfractionated mixture ( f ) and the aminoacylated fraction (p) was measured. The results were: C35, f = 0.33, p = 0.19; C36, f = 0.25, p = 0.10; C74, f = 0.28,~ = 0.26; C75, f = 0.25 ,~ = 0.20. From these data, the Modulation Constant for each reactive residue was calculated from the equation M = (f p)/ f(l p): M35 (anticodon) = 0.52; M36 (anticodon) = 0.67; M74 (CCA end) = 0.10; M75 (CCA end) = 0.25. These values, which are based on the assumptions that the C + U reactions occur independently and that the effect of each change on the acceptor activity is an independent event, express the fractional loss in activity that would occur from a C + U change at the residue in question by itself. From these results, we conclude: 1) -80% of the observed inactivation was due to changes in the anticodon; 2) neither of these anticodon residues (C35 and C36) is essential for aminoacylation; 3) a C -+ U change at C75 (CCA end) has a small effect on aminoacylation; 4) a C + U change at C74 (CCA end) has little or no effect on aminoacylation.