title: Reducing the genetic code induces massive rearrangement of the proteome.
abstract: Expanding the genetic code is an important aim of synthetic biology, but some organisms developed naturally expanded genetic codes long ago over the course of evolution. Less than 1% of all sequenced genomes encode an operon that reassigns the stop codon UAG to pyrrolysine (Pyl), a genetic code variant that results from the biosynthesis of Pyl-tRNA(Pyl). To understand the selective advantage of genetically encoding more than 20 amino acids, we constructed a markerless tRNA(Pyl) deletion strain of Methanosarcina acetivorans (ΔpylT) that cannot decode UAG as Pyl or grow on trimethylamine. Phenotypic defects in the ΔpylT strain were evident in minimal medium containing methanol. Proteomic analyses of wild type (WT) M. acetivorans and ΔpylT cells identified 841 proteins from >7,000 significant peptides detected by MS/MS. Protein production from UAG-containing mRNAs was verified for 19 proteins. Translation of UAG codons was verified by MS/MS for eight proteins, including identification of a Pyl residue in PylB, which catalyzes the first step of Pyl biosynthesis. Deletion of tRNA(Pyl) globally altered the proteome, leading to >300 differentially abundant proteins. Reduction of the genetic code from 21 to 20 amino acids led to significant down-regulation in translation initiation factors, amino acid metabolism, and methanogenesis from methanol, which was offset by a compensatory (100-fold) up-regulation in dimethyl sulfide metabolic enzymes. The data show how a natural proteome adapts to genetic code reduction and indicate that the selective value of an expanded genetic code is related to carbon source range and metabolic efficiency.
authors.affiliation: Departments of Biochemistry and Chemistry, The University of Western Ontario, London, ON N6A 5C1, Canada;
authors.forename: Patrick
authors.initials: P
authors.lastname: O'Donoghue
authors.affiliation: Departments of Molecular Biophysics and Biochemistry.
authors.forename: Laure
authors.initials: L
authors.lastname: Prat
authors.affiliation: Department of Microbiology, Technical University of Braunschweig, Braunschweig 38106, Germany; and.
authors.forename: Martin
authors.initials: M
authors.lastname: Kucklick
authors.affiliation: Departments of Molecular Biophysics and Biochemistry.
authors.forename: Johannes G
authors.initials: JG
authors.lastname: Schäfer
authors.affiliation: Division of Microbial Physiology and Molecular Biology, University of Greifswald, Greifswald 17487, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Riedel
authors.affiliation: Cellular and Molecular Physiology, and Systems Biology Institute, Yale University, New Haven, CT 06520;
authors.forename: Jesse
authors.initials: J
authors.lastname: Rinehart
authors.affiliation: Departments of Molecular Biophysics and Biochemistry, Chemistry, and dieter.soll@yale.edu ilka.heinemann@uwo.ca.
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
authors.affiliation: Departments of Biochemistry and dieter.soll@yale.edu ilka.heinemann@uwo.ca.
authors.forename: Ilka U
authors.initials: IU
authors.lastname: Heinemann
mesh_terms: D000222:Adaptation, Physiological / Q000235:genetics; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D002853:Chromatography, Liquid; D018388:Codon, Terminator / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D005815:Genetic Code; D008239:Lysine / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D017020:Methanosarcina / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D008744:Methylamines / Q000378:metabolism; D009154:Mutation; D014176:Protein Biosynthesis / Q000235:genetics; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D012345:RNA, Transfer, Amino Acid-Specific / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D019843:Archaeal Proteins; D018388:Codon, Terminator; D008744:Methylamines; D020543:Proteome; D012345:RNA, Transfer, Amino Acid-Specific; C456839:pyrrolysine; D008239:Lysine; C023336:trimethylamine
keywords: evolution; genetic code expansion; methanogenesis; pyrrolysine; tRNAPyl