Tertiary structure base pairs between D- and TpsiC-loops of Escherichia coli tRNA(Leu) play important roles in both aminoacylation and editing

Abstract

To ensure the fidelity of protein biosynthesis, aminoacyl-tRNA synthetases (aaRSs) must recognize the tRNA identity elements of their cognate tRNAs and discriminate their cognate amino acids from structurally similar ones through a proofreading (editing) reaction. For a better understanding of these processes, we investigated the role of tRNA(Leu) tertiary structure in the aminoacylation and editing reactions catalyzed by leucyl-tRNA synthetase (LeuRS). We constructed a series of Escherichia coli tRNA(Leu) mutated transcripts with alterations of the nucleotides involved in tertiary interactions. Our results revealed that any disturbance of the tertiary interaction between the tRNA(Leu) D- and TpsiC-loops affected both its aminoacylation ability and its ability to stimulate the editing reaction. Moreover, we found that the various tertiary interactions between the D- and TpsiC-loops (G18:U55, G19:C56 and U54:A58) functioned differently within the aminoacylation and editing reactions. In these two reactions, the role of base pair 19:56 was closely correlated and dependent on the hydrogen bond number. In contrast, U54:A58 was more important in aminoacylation than in editing. Taken together, our results suggest that the elbow region of tRNA formed by the tertiary interactions between the D- and TpsiC-loops affects the interactions between tRNA and aaRS effectively both in aminoacylation and in editing.

Figure 1

The structure of tRNA^Leu. (A) The L-shaped tertiary structure based on tRNA^Phe. Dotted lines indicate the tertiary interactions. (B) The cloverleaf structure of transcripts and the mutants used in this study. Dotted arrows indicate the deletion mutations, Δ16U/17U, Δ18G/19G and Δ54U/55U. Asterisks indicate the double deletion mutation (Δ18G/19G–Δ54U/55U), triangles indicate the double substitution mutation (U19:A56) and diamonds indicate the double substitution mutation (C54:G58).

Figure 2

Proposed hydrogen bonding in the tertiary base pairs. (A) G18:U55. (B) A18:U55. (C) G18:A55. (D) G19:C56. (E) U19:C56. (F) G19:A56. (G) U19:A56. (H) U54:A58. (I) A54:A58. (J) C54:A58. (K) U54:G58. (L) C54:G58. Predicted hydrogen bonds are shown as dotted lines. Nitrogen atoms are shown as solid circles. R⁺ indicates that the ribose–phosphate chain is coming toward the reader, while R^– indicates that it is facing away from the reader.

Figure 3

The misaminoacylation of tRNA transcripts by E.coli LeuRS with isoleucine.

Figure 4

tRNA^Leu-dependent stimulation of ATP hydrolysis by E.coli LeuRS in the presence of norvaline. All the data were corrected against background ATP hydrolysis.

Figure 5

Combined comparison of the activities of tRNA^Leu mutants correlated with the substitution mutation at G18:U55, G19:C56 and U54:A58 in aminoacylation and editing reactions. The aminoacylation activity is the relative k_cat/K_m, and the editing activity is the relative ATP hydrolysis rate.