Methane production was the original impetus for research into methanogenic bacteria. The innate vulnerability of current energy supplies underlies the need for their continued study. Methanogens also occupy a crucial ecological niche, preventing carbon accumulation in their anoxic environments. Prosaic but pragmatic processes such as sewage digestion rely heavily on methanogens, and many toxic chemicals are degraded under methanogenic conditions. Methylamines are preferred substrates of methanogens. Methyl-CoM is the penultimate intermediate to methane. This laboratory has biochemically reconstituted the pathways of CoM methylation from trimethylamine (TMA) and monomethylamine (MMA) in the methylotrophic methanogen Methanosarcina barkeri. These pathways share an identical CoM methylase protein, which interacts with two different, but homologous, corrinoid binding proteins specific for either TMA or MMA. The key enzymes of each pathway initiate metabolism by specifically methylating the corrinoid proteins with MMA or TMA. The TMA specific methyltransferase (TMAMT) is encoded by mttB. The MMA specific methyltransferase (MMAMT) is encoded by mtmB. These functionally analogous enzymes share little sequence similarity, except that both genes contain in-frame UAG codons. If used as a stop codon, the in-frame UAG would result in much shorter proteins than observed for the isolated methyltransferases. The UAG codons are the single interruptions of otherwise open reading frames of proper size for both proteins. For mtmB, the sequence, reading frame, and gene assignment have been exhaustively confirmed. The ability of UAG to direct transcriptional termination is clearly circumvented during synthesis of the methyltransferases. Both transcripts are predicted to have conserved structure and sequence elements around the UAG codon. UAG is used at unusually low frequency as a stop codon in M. barkeri. It appears that during the acquisition of the ability to convert methylamines to methane Methanosarcina also acquired a violation of the canonical genetic code. A number of hypothetical means of preventing termination at UAG are possible and will be tested. Peptide maps of MMAMT will be generated and the protein sequence of peptides encoded by the UAG-containing regions will be determined by mass spectroscopy and Edman degradation. Any amino acid encoded by UAG will be characterized. Preliminary data indicates UAG readthrough is a translational event, but transcripts will be further examined for evidence of processing or editing. The efficiency of UAG readthrough will be tested under different growth conditions using antibody to the full-length protein. Divergent mtmB and mttB genes from related genera will be compared to ascertain conservation of the UAG codon, its position, and surrounding sequence/structure context. Reporter fusions with 5' UAG containing regions will be introduced into the chromosome in order to determine effects of sequence context and growth conditions on UAG readthrough. An in vitro translation system will also be used to test the effect of sequence context and growth conditions on UAG readthrough in constructs ranging from short messages with single in-frame stops to full length mtmB containing transcripts. Even if the current model for how suppression of UAG directed termination occurs is misleading, there will remain evidence for the first example of an intron in protein coding genes in the Archaea, an entity very seldom seen among any prokaryote. The potential to learn something new here is high. No matter what the mechanism for circumventing UAG-directed termination may turn out to be, this system offers rare insight into how organisms evolve new metabolic capabilities, even to the extent of modifying their genetic codes.
