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. 2000 Aug 1;97(16):8916-20.
doi: 10.1073/pnas.97.16.8916.

Hydrolytic editing by a class II aminoacyl-tRNA synthetase

P J Beuning  1 , K Musier-Forsyth
Affiliations

Hydrolytic editing by a class II aminoacyl-tRNA synthetase

P J Beuning et al. Proc Natl Acad Sci U S A. .

Abstract

Editing reactions catalyzed by aminoacyl-tRNA synthetases are critical for accurate translation of the genetic code. To date, this activity, whereby misactivated amino acids are hydrolyzed either before or after transfer to noncognate tRNAs, has been characterized extensively only in the case of class I synthetases. Class II synthetases have an active-site architecture that is completely distinct from that of class I. Thus, findings on editing by class I synthetases may not be applicable generally to class II enzymes. Class II Escherichia coli proline-tRNA synthetase is shown here to misactivate alanine and to hydrolyze the noncognate amino acid before transfer to tRNA(Pro). This enzyme also is capable of rapidly deacylating a mischarged Ala-tRNA(Pro) variant. A single cysteine residue (C443) that is located within the class II-specific motif 3 consensus sequence was shown previously to be dispensable for proline-tRNA synthetase aminoacylation activity. We show here that C443 is critical for the hydrolytic editing of Ala-tRNA(Pro) by this class II synthetase.

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Figures

Figure 1
Figure 1
Noncognate amino acids stimulate ATP hydrolysis by E. coli ProRS. Graph showing the ATP hydrolysis activity of ProRS (2 μM) in the presence of 250 mM trans-4-hydroxyproline (trans) (⧫), 500 mM alanine (▴), 250 mM cis-4-hydroxyproline (cis) (▾), 250 mM azetidine-4-carboxylic acid (azetidine) (●), and 2 mM proline (■).
Figure 2
Figure 2
Deacylation of wild-type tRNAPro and mischarged tRNAPro variants. (A) The sequence of alanine-accepting E. coli tRNAPro. The shaded boxes indicate nucleotides that are mutated relative to wild-type E. coli tRNAPro. (B–D) Graphs showing the deacylation of G1:C72/U70-[3H]Ala-tRNAPro (B), E. coli [3H]Pro-tRNAPro (C), and human [3H]Pro-tRNAPro (D) in the presence (+) and absence (−) of E. coli ProRS. (E) Graph showing that the efficient deacylation of the Ala-tRNAPro variant is specific for E. coli (Ec) ProRS (0.5 μM). E. coli AlaRS (1 μM) (⧫), human (Hs) ProRS (0.6 μM) (●), and M. jannaschii (Mj) ProRS (0.4 μM) (▾) were unable to deacylate E. coli G1:C72/U70-Ala-tRNAPro. (F) G1:C72/U70-[3H]Lys-tRNAPro is not deacylated by E. coli ProRS. In B-–F, the data obtained in the presence of E. coli ProRS are represented by ■, and the control reaction carried out in the absence of enzyme is represented by ▴.
Figure 3
Figure 3
Deacylation of G1:C72/U70-Ala-tRNAPro by wild-type (WT) and C443G ProRS. (A) Deacylation by 0.1 μM C443G-ProRS (■) and wild-type ProRS (●). (B) Deacylation by 0.5 μM (■), 1 μM (▴), and 2 μM (▾) C443G-ProRS. In both graphs, an assay performed in the absence of ProRS (⧫) is shown.
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