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Pyrrolysine is a lysine derivative encoded by the UAG codon in methylamine methyltransferase genes of Methanosarcina barkeri. Near a methyltransferase gene cluster is the pyIT gene, which encodes an unusual transfer RNA (tRNA) with a CUA anticodon. The adjacent pyIS gene encodes a class II aminoacyltRNA synthetase that charges the pyIT-derived tRNA with lysine but is not closely related to known lysyl-tRNA synthetases. Homologs of pyIS and pyIT are found in a Gram-positive bacterium. Charging a tRNA^sub cua^ with lysine is a likely first step in translating UAG amber codons as pyrrolysine in certain methanogens. Our results indicate that pyrrolysine is the 22nd genetically encoded natural amino acid.
In Methanosarcina species, specific methyltransferases initiate methanogenesis and carbon assimilation from substrates such as trimethylamine (TMA), dimethylamine (DMA), or monomethylamine (MMA). The highly expressed, nonhomologous genes encoding these methyltransferases have inframe UAG (amber) codons that do not stop translation during synthesis of the full-length proteins (1-3). Nearly identical copies of the DMA and MMA methyltransferase genes with conserved single in-frame amber codons occur in the same genome (2, 4). There is no evidence for transcript editing (2, 4), and, unlike many other stop codon readthrough events (5), readthrough of the amber codons is highly efficient (3). Amber serves as a sense codon within the methylamine methyltransferase genes (3), previously unknown in any other group of archaeal genes. In the accompanying manuscript, Hao et al. have shown that in intact MtmB the amber-encoded residue is pyrrolysine, whose structure is proposed as lysine with its epsilon nitrogen in amide linkage with (4R,SR)4-substitutedpyrroline-5-carboxylate. Here, we describe a specialized tRNA,,AU" and lysyl-tRNA synthetase (LysRS) that underlie amber codon translation as pyrrolysine in certain methane-- producing Archaea.
An unannotated gene, pylT (Fig. 1), whose predicted tRNA product has a CUA anticodon, was identified in the Methanosarcina barkeri Fusaro genomic database (GenBank accession number NC_002724) using tRNAScanSE (6, 7). Sequencing of M. barkeri MS DNA (7) also revealed pyIT, as well as the three following open reading frames, pyIS, pyIB, and pyIC (GenBank AY064401). Northern blots of the RNA pool from MMA-grown M. barkeri MS revealed an RNA of the size expected for the tRNA^sub CUA^ product of pyIT (Fig. 1C). The predicted secondary structure of tRNA^sub CUA^ has unusual properties compared...