In vitro study of E.coli tRNA(Arg) and tRNA(Lys) identity elements

Abstract

Various tRNA transcripts were constructed to study the identity elements of E.coli tRNA(Arg) and tRNA(Lys). Exchange of the anticodon of the major tRNA(Arg) from ACG to either CCG or CCU did not result in a significant loss of arginine acceptor activity, whereas not only that to UUU but also that to ACA or ACC decreased the activity. Base substitutions and deletion at A20 also impaired the arginine charging activity by over 50-fold. Arginine charging activity was introduced by either substitution of the anticodon from UAC to ACG in tRNA(Val) or from UUU to UCU in tRNA(Lys). Only a single base substitution at the third position of tRNA(Trp) anticodon (CCA) from A to G also gave rise to arginine charging activity, which was elevated to a comparable level to that of the tRNA(Arg) transcript by an additional A20 insertion. Base substitutions of the major tRNA(Arg) at the discriminator position into pyrimidines led to a decrease by factors of three to four. These data show that the third letter of the anticodon G36 or U36 besides the second letter C35 and the A20 in the variable pocket is responsible for the arginine acceptor identity, to which the discriminator base A73 or G73 contributes in an auxiliary fashion. In contrast to the arginine system, the transcript with the wild-type tRNA(Lys) sequence showed only 140-fold lower lysine charging activity than the native tRNA(Lys), suggesting the involvement of base modifications in recognition. Replacement of the anticodon UUU with not only UCU and UAC but also UUA and UUC seriously affected the lysine acceptor activity, and those with GUU and UUG also decreased by factors of 17 and 5, respectively. Introduction of UUU into the anticodons conferred lysine charging activity upon both tRNA(Val) and tRNA(Arg). Substitution of the discriminator base A73 by any of the other bases decreased the lysine acceptor activity by a factor of ten. These results indicate the involvements of all the three bases of the anticodon and A at the discriminator position in lysine specific aminoacylation.