The appearance of pyrrolysine in tRNAHis guanylyltransferase by neutral evolution

Abstract

tRNA(His) guanylyltransferase (Thg1) post-transcriptionally adds a G (position -1) to the 5'-terminus of tRNA(His). The Methanosarcina acetivorans Thg1 (MaThg1) gene contains an in-frame TAG (amber) codon. Although a UAG codon typically directs translation termination, its presence in Methanosarcina mRNA may lead to pyrrolysine (Pyl) incorporation achieved by Pyl-tRNA(Pyl), the product of pyrrolysyl-tRNA synthetase. Sequencing of the MaThg1 gene and transcript confirmed the amber codon. Translation of MaThg1 mRNA led to a full-length, Pyl-containing, active enzyme as determined by immunoblotting, mass spectrometry, and biochemical analysis. The nature of the inserted amino acid at the position specified by UAG is not critical, as Pyl or Trp insertion yields active MaThg1 variants in M. acetivorans and equal amounts of full-length protein. These data suggest that Pyl insertion is akin to natural suppression and unlike the active stop codon reassignment that is required for selenocysteine insertion. Only three Pyl-containing proteins have been characterized previously, a set of methylamine methyltransferases in which Pyl is assumed to have specifically evolved to be a key active-site constituent. In contrast, Pyl in MaThg1 is a dispensable residue that appears to confer no selective advantage. Phylogenetic analysis suggests that Thg1 is becoming dispensable in the archaea, and furthermore supports the hypothesis that Pyl appeared in MaThg1 as the result of neutral evolution. This indicates that even the most unusual amino acid can play an ordinary role in proteins.

Fig. 1.

Encoding of Pyl in MaThg1. Sequences are displayed in green for peptide MA0816 and in red for MA0817. The amber stop codon TAG and the downstream Met codon ATG are marked in bold. The Pyl containing peptide linker and its coding sequence between MA0817 and MA0816 is shown in black. (A) Sequencing of genomic DNA (gDNA) and cDNA confirmed in-frame amber codon in the thg1 gene. (B) A secondary structure prediction of MaThg1 places the Pyl residue within an α-helix.

Fig. 2.

UAG translation in Thg1 in M. acetivorans. (A) SDS-solubilized MaThg1 mutant Pyl142Trp purified from E. coli (rTrp) and extracts of M. acetivorans expressing His-tagged Thg1 (Pyl) were analyzed by anti-His immunoblot. (B) Deconvolution of Thg1 protein purified from M. acetivorans analyzed by ESI-TOF MS. (C) MS/MS spectrum of peptide 137–148 with a Pyl at position 142 (m/z of 500.5³⁺). The location where the precursor ion is indicated by a solid diamond. Observed fragment ions (b and y) were consistent with the following McLafferty diagram: N F/V A S/O G Y Y/A/L/R where O indicates a Pyl residue.

Fig. 3.

Role of Pyl in MaThg1. (A) SDS-solubilized extracts of M. acetivorans expressing His-tagged Thg1 (Pyl) and His-tagged Thg1 mutant Pyl142Trp (Trp) grown on either methanol (MeOH) or TMA were analyzed by anti-His immunoblot. M. acetivorans C2A cell extracts were used as control (−). (B) Recombinant MaThg1 mutant Pyl142Trp (rTrp) and the two Thg1 halves MA0817 (r1) and MA0816 (r2) were produced and purified from E. coli. The Pyl containing MaThg1 (Pyl) variant was purified from M. acetivorans. MA0817 and MA0816 were assayed separately and together (r1+r2).

Fig. 4.

Enzymatic activity and substrate specificity of MaThg1. (A) M. acetivorans tRNA^His. Residues conserved among M. thermautotrophicus, P. aerophilum and E. coli tRNA^His are marked in blue. The G-1 residue is highlighted. (B) Activity of Thg1 was assayed by addition of M. acetivorans tRNA^HisΔG-1 and [α-³²P]GTP to cell-free extracts from M. acetivorans grown on TMA or methanol (MeOH). tRNA^HisΔG-1 from P. aerophilum (78 nt), M. thermautotrophicus (75 nt), E. coli (75 nt), and from M. acetivorans (75 nt) were assayed with Thg1 purified from M. acetivorans. (C) Sequence alignment of tRNA^His from M. thermautotrophicus, E. coli, P. aerophilum, and M. acetivorans.

Fig. 5.

Phylogeny of archaeal Thg1. All archaea known to encode Thg1 are included in the maximum likelihood phylogeny (A). Magnification (B) of the Methanosarcinaceae Thg1 phylogenetic pattern (gray box in A). The codon and amino acid at the position homologous to Pyl142 in MaThg1 are shown adjacent to the branches. The most parsimonious sequence changes at position 142 are shown beneath. Bootstrap support is given at each node.