leucylation, because all tRNA Ser CAGs possessing leucyl-In order to verify that the leucylation activity observed ation activity have m 1 G in common, while only the for the tRNA Ser CAG of C.zeylanoides actually came from tRNA Ser CAG of C.cylindracea, which possesses no leucylthe tRNA Ser CAG itself, and not from a trace amount ation activity, has A at this position. of leucine tRNA contaminating the tRNA sample, the To examine the validity of this speculation, a series of leucylated 3Ј-terminal RNA fragment derived from leucyl-tRNA Ser CAG variants was constructed by the in vitro tRNA Ser CAG was analyzed in the following manner. 14 Ctranscription method using T7 RNA polymerase, as well leucylated tRNA Ser CAG from C.zeylanoides was first as by the microsurgery method, and the leucylation activity acetylated with acetic anhydride to prevent deacylation, of each variant was measured. When the tRNA Ser CAG of and then digested with RNase T1. The resulting 3Ј-C.zeylanoides synthesized by in vitro transcription was terminal fragment with 14 C-labeled acetylleucine was employed as a substrate, no leucylation activity was analyzed by cellulose TLC. The results are shown in detected, not even for the tRNA transcript having G37 Figure 1C . If leucylated tRNA Ser CAG were digested ( Figure 3A ). On the other hand, as shown in Figure 3A , with RNase T1, 14 C-labeled acetylleucyl-CCA should be serylation activity exceeded 1000 pmol/A 260 unit. These released as a labeled fragment ( Figure 1C , lane 3), because results strongly suggested that some nucleoside modifica-G is located at position 73 of the tRNA Ser CAG (Figure tion is necessary in tRNA Ser CAG for recognition by 1A, left-hand structure). Any contaminated leucine tRNAs, LeuRS. We thus attempted to replace the m 1 G37 of if they exist, will give some 14 C-labeled fragments larger C.zeylanoides tRNA Ser CAG with G (the variant is symbolthan the tetramer ( Figure 1C , lane 4), because all the ized as m 1 G37G) or A (m 1 G37A), by the microsurgery leucine tRNAs of yeasts so far analyzed (Sprinzl et al., method (Figure 2A and B; for details, see Materials 1996) including those of C.zeylanoides (T. Suzuki, unpuband methods) to examine the contribution of m 1 G37 to lished result) are known to have A73 at their 3Ј-ends, leucylation and the contribution of A37 of C.cylindracea which are resistant to RNase T1. The mobility of the tRNA Ser CAG to the prevention of leucylation. acetylleucyl-oligonucleotide derived from tRNA Ser CAG When aminoacylation of m 1 G37A and m 1 G37G was from C.zeylanoides ( Figure 1C , lane 1) was identical to examined ( Figure 3A ), the results indicated that both that of acelylleucyl-CCA prepared from the RNase U2 substitutions lead to complete loss of leucylation (Figure digests of leucyl-tRNA Leu s from C.zeylanoides (lane 3).