The effect of bisulfite-induced C to U transitions on aminoacylation of Escherichia coli glycine tRNA.
The effect of bisulfite-induced C to U transitions on the aminoacylation of highly purified Escherichia coli tRNAgly3,gcc has been studied. On treatment with 3.2 M NaHSO3 at 25 degrees C and pH 5.8, C to 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 tRNAgly3,gcc exists in the native conformation under the conditions of the reaction; 2) its ordered structure is similar to yeast tRNAPhe; 3) its anticodon loop is flexible in solution. During the reaction, loss of glycine acceptor activity followed first order kinetics with a t1/2 = 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, p = 0.26; C75, f = 0.25, p = 0.20. From these data, the Modulation Constant for each reactive residue was calculated from the equation M = (f - p)/f(1 - 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 to 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 to U change at the residue in question by itself. From these results, we conclude: 1) approximately 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 to U change at C75 (CCA end) has a small effect on aminoacylation; 4) a C to U change at C74 (CCA end) has little or no effect on aminoacylation.
D003596:Cytosine; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics*; D013447:Sulfites / Q000494:pharmacology*; D014498:Uracil
D012346:RNA, Transfer, Amino Acyl; D013447:Sulfites; D014498:Uracil; D003596:Cytosine