Volume 19, Issue 2, Pages 235-246 (July 2005) Molecular Mechanism of Lysidine Synthesis that Determines tRNA Identity and Codon Recognition  Yoshiho Ikeuchi, Akiko Soma, Tomotake Ote, Jun-ichi Kato, Yasuhiko Sekine, Tsutomu Suzuki  Molecular Cell  Volume 19, Issue 2, Pages 235-246 (July 2005) DOI: 10.1016/j.molcel.2005.06.007 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 ATP Hydrolysis and an Adenylate Intermediate in Lysidine Formation (A) TLC analysis of the hydrolysis product of ATP. γ-32P-labeled (lanes 1 and 2) and α-32P-labeled (lanes 3 and 4) ATP were hydrolyzed by TilS during in vitro lysidine formation. Aliquots (5000 cpm) of the reaction mixture before (lanes 1 and 3) and after (lanes 2 and 4) lysidine formation were subjected to PEI cellulose TLC. Positions for AMP, PPi, and ATP are indicated. (B) Detection by PAGE of an adenylate intermediate during lysidine formation. The upper and lower panels show ethidium bromide staining and visualized radioactivity, respectively. Lysidine formation of tRNAIle2 (C34) in the presence of [α-32P]ATP without Lys (lane 1), with Lys (lane 2), or with Arg (lane 3). The radioactivity of the AMP bound tRNA can be seen in lanes 1 and 3 of the lower panel. A tRNAIle2 with G34 treated without Lys (lane 4) and with Lys (lane 5) served as a negative control. (C) Proposed two-step lysidine formation reaction. TilS activates the C-2 position of C34 in tRNAIle2 to form an adenylate intermediate and release PPi. The ϵ-amino group of lysine attacks this adenylate to release AMP and complete the lysidine formation. Molecular Cell 2005 19, 235-246DOI: (10.1016/j.molcel.2005.06.007) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Domain Structure of TilS and Introduced Mutations (A) Schematic depiction of the domain structure of the TilS protein. NTD and CTD stand for N-terminal and C-terminal domain, respectively. The conserved motifs and mutated sites are indicated. (B) Ribbon model of E. coli TilS (1NI5). Amino acids that were mutated in this study are indicated. (C) Acid-urea PAGE combined with Northern analysis of tRNAIle2 from tilSts strains. Upper and lower bands represent tRNAIle2 with L34 and C34 (ΔL), respectively. The ratio of the quantified upper and lower bands represents the L content (%). Each strain indicated at the top of the gel is listed in Table S2. (D) Lysidine formation in vitro by A. aeolicus TilS. Lysidinylation of A. aeolicus tRNAIle transcript (closed circle) and E. coli tRNAIle transcript (closed square) by A. aeolicus TilS is shown. Lysidinylation of E. coli tRNAIle transcript (open circle) and A. aeolicus tRNAIle transcript (open square) by E. coli TilS is shown. Molecular Cell 2005 19, 235-246DOI: (10.1016/j.molcel.2005.06.007) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Mutation Study of tRNAs to Investigate Positive and Negative Determinants for Lysidine Formation (A and B) tRNA variants based on E. coli tRNAIle2 (A) and E. coli elongator tRNAMet (B) that were used in this study. The numbering system of the tRNA is based on the tRNA compilation by Sprinzl and Vassilenko (2005). The arrows indicate the substitutions and insertions made in this study. The kinetic parameters for these tRNA variants are summarized in Table 2 (tRNAIle2) and Table 3 (tRNAMet). (C) Positive determinants for TilS identified by the mutation study are boxed in the secondary structure of tRNAIle2. Identity elements (positive determinants) for IleRS are shown in blue. The anticodon (positions 34–36), the discriminator base (A73), and the base pairs G2-C71 and C3-G70 also act as identity elements for MetRS. Phosphate positions protected by TilS in the footprint experiment are indicated by arrows. (D) Negative determinants for TilS identified by the mutation study are encircled in the secondary structure of tRNAMet. Identity elements (positive determinants) for MetRS are shown in red. (E) Secondary structure of A. aeolicus tRNAIle without modifications. The encircled G5-C68 base pair works as a negative determinant for E. coli TilS. Molecular Cell 2005 19, 235-246DOI: (10.1016/j.molcel.2005.06.007) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Identification of TilS Binding Sites on tRNAIle2 by Footprinting Analysis and the Proposed Binding Model (A) A footprinting experiment was performed on 5′ end-labeled tRNA with (lanes 4–6) or without (lanes 1–3) ENU. The experiment was performed in the absence (lanes 1 and 4) or presence (lanes 2 and 5) of TilS, or in the presence of BSA (lanes 3 and 6). The products of partial digestion by RNase T1 (lane T1) and alkaline treatment (lane Al) were electrophoresed to form molecular size ladders. White lines on the right side of lane 5 indicate the protected sites. (B) Schematic depiction of a model of the tRNAIle2-TilS complex. In the simplified structure of TilS, a hole in the NTD is indicated as a cylindrical shape. The P loop is shown in red. A depiction of tRNAIle2 indicating the TilS binding sites is shown. In the proposed binding model, upon tRNA binding, the NTD changes its conformation, which expands the hole in the NTD. ATP and lysine may enter the active center through the bottom hole of NTD, and PPi and AMP may be released through the same hole. Molecular Cell 2005 19, 235-246DOI: (10.1016/j.molcel.2005.06.007) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Network of Determinants that Guide tRNA Modification and Aminoacylation Three sets of positive determinants for TilS, MetRS, and IleRS and one set of negative determinants for TmcA are embedded in the primary sequence of tRNAIle2. In contrast, tRNAMet has two sets of positive determinants for MetRS and TmcA and two sets of negative determinants for IleRS and TilS. Thus, determinants that guide the introduction of wobble modifications control the amino acid specificities of the tRNAs. Molecular Cell 2005 19, 235-246DOI: (10.1016/j.molcel.2005.06.007) Copyright © 2005 Elsevier Inc. Terms and Conditions