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. 2003 Jun 1;31(11):2865-72.
doi: 10.1093/nar/gkg382.

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

Xing Du  1 , En-Duo Wang
Affiliations

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

Xing Du et al. Nucleic Acids Res. .

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.

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Figures

Figure 1
Figure 1
The structure of tRNALeu. (A) The L-shaped tertiary structure based on tRNAPhe. 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
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
Figure 3
The misaminoacylation of tRNA transcripts by E.coli LeuRS with isoleucine.
Figure 4
Figure 4
tRNALeu-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
Figure 5
Combined comparison of the activities of tRNALeu 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 kcat/Km, and the editing activity is the relative ATP hydrolysis rate.
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