The amber codon in the gene encoding the monomethylamine methyltransferase isolated from Methanosarcina barkeri is translated as a sense codon.
Each of the genes encoding the methyltransferases initiating methanogenesis from trimethylamine, dimethylamine, or monomethylamine by various Methanosarcina species possesses one naturally occurring in-frame amber codon that does not appear to act as a translation stop during synthesis of the biochemically characterized methyltransferase. To investigate the means by which suppression of the amber codon within these genes occurs, MtmB, a methyltransferase initiating metabolism of monomethylamine, was examined. The C-terminal sequence of MtmB indicated that synthesis of this mtmB1 gene product did not cease at the internal amber codon, but at the following ochre codon. Antibody raised against MtmB revealed that Escherichia coli transformed with mtmB1 produced the amber termination product. The same antibody detected primarily a 50-kDa protein in Methanosarcina barkeri, which is the mass predicted for the amber readthrough product of the mtmB1 gene. Sequencing of peptide fragments from MtmB by Edman degradation and mass spectrometry revealed no change in the reading frame during mtmB1 expression. The amber codon position corresponded to a lysyl residue using either sequencing technique. The amber codon is thus read through during translation at apparently high efficiency and corresponds to lysine in tryptic fragments of MtmB even though canonical lysine codon usage is encountered in other Methanosarcina genes.
James
C M
CM
Department of Microbiology, Ohio State University, Columbus, Ohio 43210, USA.
Ferguson
T K
TK
Leykam
J F
JF
Krzycki
J A
JA
D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D002851:Chromatography, High Pressure Liquid; D003062:Codon; D018388:Codon, Terminator; D017931:DNA Primers / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015151:Immunoblotting; D013058:Mass Spectrometry; D017020:Methanosarcina / Q000201:enzymology*; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology
D019843:Archaeal Proteins; D003062:Codon; D018388:Codon, Terminator; D017931:DNA Primers; D008780:Methyltransferases; C113200:monomethylamine methyltransferase; D014357:Trypsin