Pyrrolysine is the 22 super(nd) genetically encoded amino acid to be found in nature. Co-translational insertion at in-frame UAG codons proceeds so that a single pyrrolysine residue is found in the active site of all methylamine methyltransferases required for methylamine metabolism in Methanosarcina spp. This study examines processes central to the translation of UAG as pyrrolysine. The pylT gene encoding the tRNA for pyrrolysine, tRNA super( Pyl), is part of the pyl operon of Methanosarcina spp. which also contains the pylS gene, encoding a class II aminoacyl-tRNA synthetase; and the pylB, pylC, and pylD genes proposed to catalyze the synthesis of pyrrolysine from metabolic precursors. Here, in the first study, by characterizing a Methanosarcina acetivorans mutant with the pylT gene region deleted, the essentiality of pyrrolysine incorporation in methanogenesis from methylamines is tested. The mutant lacks detectable tRNA super(Pyl) but grows similar to wild-type on methanol or acetate for which translation of UAG as pyrrolysine is likely not to be essential. However, unlike wild-type, the mutant can not grow on any methylamine or use monomethylamine as the sole source of nitrogen. Monomethylamine methyltransferase activity is detectable in wild-type cells, but not in mutant cells during growth on methanol. Further, the pyrrolysine-containing monomethylamine methyltransferase is absent from the mutant. This study is the first genetic analysis of UAG translation as pyrrolysine, and it reveals the phenotype of a Methanosarcina strain that can not decode UAG as pyrrolysine. PylS is an aminoacyl-tRNA synthetase encoded by the pylS gene adjacent to pylT. The data presented here in the second study shows that PylS catalyzes the ATP-dependent activation of synthetic pyrrolysine. PylS-catalyzed activation is highly specific for cognate amino acid, pyrrolysine, and does not require tRNA. Taken together with results obtained by others showing the ligation of pyrrolysine to tRNA super(Pyl) and the ability of pylS and pylT gene products to suppress amber codons in a recombinant system, the data presented indicates that PylS is a pyrrolysyl-tRNA synthetase capable of directly ligating pyrrolysine to tRNA super(Pyl) in vitro and in vivo. These results prove that PylS is the first aminoacyl-tRNA synthetase to be discovered from nature that is capable of attaching a genetically-encoded amino acid, not one of the common twenty, to cognate tRNA. Further, a metabolite from an Escherichia coli strain with the pylBCD genes expressed is shown to serve as a substrate for PylS activation. Along with results obtained by others showing PylS-catalyzed ligation of this metabolite to tRNA super( Pyl), the data indicates that the pylB, pylC, and pylD gene products are sufficient for pyrrolysine synthesis from metabolic precursors common to M. acetivorans and E. coli. Earlier, in vitro studies by others indicated the two canonical lysyl-tRNA synthetases found in Methanosarcina spp. form a complex and slowly attach lysine to tRNA super(Pyl). Owing to the implicit problem of substrate selection between lysine-tRNA super( Pyl) and pyrrolysine-tRNA super(Pyl) in translation, the relevance of the complex catalyzed ligation required testing in vivo. This is the focus of the third study. Data presented indicates that intact lysyl-tRNA synthetase genes are not required for methanogenesis from methylamines in Methanosarcina. Further, levels of monomethylamine methyltransferase and tRNA super(Pyl) aminoacylation are not diminished in lysyl-tRNA synthetase mutants. Taken together, the data presented here demonstrates that the indirect route of tRNA super(Pyl) aminoacylation involving complex formation of the two lysyl-tRNA synthetases is not essential for UAG translation as pyrrolysine. In the final study, the substrate specificity of PylS is probed using analogs of pyrrolysine. Features of the pyrroline ring of pyrrolysine that are determinants of PylS catalyzed activation are identified. Analogs used in functional probing in vitro are then incorporated in a monomethylamine methyltransferase in an Escherichia coli reporter system.
Methylamine; Methanogenic archaea; Data processing; Insertion; Lysine; Operons; Amino acids; Stop codon; Methanosarcina; Codons; Acetic acid; Substrate specificity; Metabolites; Archaea; Metabolism; Methanosarcina acetivorans; tRNA; Aminoacylation; Methanogenesis; Genetic analysis; Translation; Escherichia coli; Nitrogen; Methanol; Methyltransferase; Aminoacyl-tRNA ligase