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, 101 (34), 12450-4

An aminoacyl-tRNA Synthetase That Specifically Activates Pyrrolysine

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An aminoacyl-tRNA Synthetase That Specifically Activates Pyrrolysine

Carla Polycarpo et al. Proc Natl Acad Sci U S A.

Abstract

Pyrrolysine, the 22nd cotranslationally inserted amino acid, was found in the Methanosarcina barkeri monomethylamine methyltransferase protein in a position that is encoded by an in-frame UAG stop codon in the mRNA. M. barkeri encodes a special amber suppressor tRNA (tRNA(Pyl)) that presumably recognizes this UAG codon. It was reported that Lys-tRNA(Pyl) can be formed by the aminoacyl-tRNA synthetase-like M. barkeri protein PylS [Srinivasan, G., James, C. M. & Krzycki, J. A. (2002) Science 296, 1459-1462], whereas a later article showed that Lys-tRNA(Pyl) is synthesized by the combined action of LysRS1 and LysRS2, the two different M. barkeri lysyl-tRNA synthetases. Pyrrolysyl-tRNA(Pyl) formation was presumed to result from subsequent modification of lysine attached to tRNA(Pyl). To investigate whether pyrrolysine can be directly attached to tRNA(Pyl) we chemically synthesized pyrrolysine. We show that PylS is a specialized aminoacyl-tRNA synthetase for charging pyrrolysine to tRNA(Pyl); lysine and tRNA(Lys) are not substrates of the enzyme. In view of the properties of PylS we propose to name this enzyme pyrrolysyl-tRNA synthetase. In contrast, the LysRS1:LysRS2 complex does not recognize pyrrolysine and charges tRNA(Pyl) with lysine. These in vitro data suggest that Methanosarcina cells have two pathways for acylating the suppressor tRNA(Pyl). This would ensure efficient translation of the in-frame UAG codon in case of pyrrolysine deficiency and safeguard the biosynthesis of the proteins whose genes contain this special codon.

Figures

Fig. 1.
Fig. 1.
Structures of pyrrolysine derivatives. (A and B) Diastereomers of the chemically synthesized N6-[(3-methyl-2,3-dihydro-1H-pyrrol-2-yl)carbonyl]-lysine. (C) Proposed structure of pyrrolysine. The chiral arrangement in the pyrrole ring was patterned after the crystallographic image in figure 2C of ref. .
Fig. 2.
Fig. 2.
Reconstructed ion chromatograms for m/z 256 (MH+ for pyrrolysine) from LC/ESI MS analysis of synthetic pyrrolysine (A) and the amino acid in pryrrolysyl-tRNAPyl generated by PylS in the presence of the synthetic pyrrolysine preparation (B).
Fig. 3.
Fig. 3.
Northern blot analysis of the M. barkeri Fusaro tRNAPyl transcript charged with lysine (Lys) or pyrrolysine (Pyl) by the M. barkeri Fusaro PylS, LysRS1, and LysRS2 enzymes. The resulting tRNA products were loaded onto an acid gel. After transfer to a nitrocellulose membrane, the samples were hybridized with a tRNAPyl-specific oligonucleotide probe. The positions of tRNAPyl, pyrrolysyl-tRNAPyl, and lysyl-tRNAPyl are indicated. The slower-moving band (above tRNAPyl) is an extended transcription product.
Fig. 4.
Fig. 4.
Pyrrolysyl-AMP synthesis by PylS as measured in the ATP-PPi exchange reaction. The experimental conditions are described in Materials and Methods. The reaction was conducted in the presence of either 500 μM pyrrolysine (•) or 500 μM lysine (○). Background level was determined in the absence of amino acid (▴).
Fig. 5.
Fig. 5.
Two routes of tRNAPyl aminoacylation. (Upper) PylRS acylates tRNAPyl with Pyl. (Lower) lysyl-tRNAPyl is formed by LysRS1 and LysRS2 acting together (6). Further modification of lysyl-tRNAPyl (stippled arrow) by additional uncharacterized pyl genes has been suggested as a route to pyrrolysyl-tRNAPyl (5).

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