Exceptions to the Canonical Genetic Code in a Methanogenic Archaeon

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Methanogenesis (the synthesis of methane) from methylamines is initiated in microbes by one of three methylamine methyltransferases that methylate corrinoid cofactors specifically using either trimethylamine (TMA), dimethylamine (DMA), or monomethylamine (MMA). The genes encoding the TMA, DMA, and MMA methyltransferases are non-homologous, yet each gene contains a single in-frame amber codon. If amber codons were read as stop codons, each methylamine methyltransferase gene would produce an amber termination product markedly truncated relative to the isolated and characterized methyltransferase. Sequencing of the tryptic fragments of the MMA methyltransferase revealed that readthrough occurs without a frameshift, and that the amber codon is translated as lysine in a representative methyltransferase. No editing or changes are evident in transcripts, indicating direct translation of the amber codon as lysine occurs. Two phylogenetically distinct lys-tRNA synthetase (lysRS) genes were found in Methanosarcina barkeri. A class I lysRS synthetase is found downstream of an operon with two of the amber containing methyltransferase genes and is co-regulated with the MMA methyltransferase gene. A constitutive class II synthetase is also encoded in the genome. A possible role of the class I enzyme is charging an amber lys-tRNA for translation of amber as lysine. Working hypotheses are that the amber codons are required for either proper folding and/or translational processing of the methyltransferase proteins, or are involved in controlling methyltransferase transcription and translation. The regulation of amber codon readthrough, as well as cis requirements for amber translation, will be examined using either an in vitro amber readthrough assay, or alternatively, using in vivo reporters of amber translation. A mutant strain will be constructed in which the amber codon of a methylamine methyltransferase gene is changed to a canonical lysine codon, and the effect on transcripts, protein levels and enzyme activity determined. The amber lys-tRNA will be identified by one of several alternate approaches and characterized. The two lysRS enzymes will either be expressed recombinantly or isolated from the native host in order to characterize their behavior with respect to lysine and different lys-tRNAs. These studies will shed light on the function of a change in the usual genetic code found in these methane producing microorganisms. In a select group of their genes a signal that usually stops biosynthesis of a protein, instead directs the insertion of an amino acid into the protein. Since the genetic code is universal such changes in the genetic code are relatively rare. This research project will in part explain the biological driving force behind this change in the genetic code. This is expected to provide new insights into the process of protein biosynthesis, gene regulation, and the catalytic activities of enzymes in methane-producing microbes. Understanding these processes is important because methanogenesis is an essential component of the global carbon cycle.