title: Multiple Quality Control Pathways Limit Non-protein Amino Acid Use by Yeast Cytoplasmic Phenylalanyl-tRNA Synthetase.
abstract: Non-protein amino acids, particularly isomers of the proteinogenic amino acids, present a threat to proteome integrity if they are mistakenly inserted into proteins. Quality control during aminoacyl-tRNA synthesis reduces non-protein amino acid incorporation by both substrate discrimination and proofreading. For example phenylalanyl-tRNA synthetase (PheRS) proofreads the non-protein hydroxylated phenylalanine derivative m-Tyr after its attachment to tRNA(Phe) We now show in Saccharomyces cerevisiae that PheRS misacylation of tRNA(Phe) with the more abundant Phe oxidation product o-Tyr is limited by kinetic discrimination against o-Tyr-AMP in the transfer step followed by o-Tyr-AMP release from the synthetic active site. This selective rejection of a non-protein aminoacyl-adenylate is in addition to known kinetic discrimination against certain non-cognates in the activation step as well as catalytic hydrolysis of mispaired aminoacyl-tRNA(Phe) species. We also report an unexpected resistance to cytotoxicity by a S. cerevisiae mutant with ablated post-transfer editing activity when supplemented with o-Tyr, cognate Phe, or Ala, the latter of which is not a substrate for activation by this enzyme. Our phenotypic, metabolomic, and kinetic analyses indicate at least three modes of discrimination against non-protein amino acids by S. cerevisiae PheRS and support a non-canonical role for SccytoPheRS post-transfer editing in response to amino acid stress.
authors.affiliation: From the Ohio State Biochemistry Program, Department of Microbiology, The Ohio State University, Columbus, Ohio 43210 and.
authors.forename: Adil
authors.initials: A
authors.lastname: Moghal
authors.affiliation: Pasarow Mass Spectrometry Laboratory, Semel Institute of Neuroscience and Human Behavior, University of California, Los Angeles, California 90095.
authors.forename: Lin
authors.initials: L
authors.lastname: Hwang
authors.affiliation: Pasarow Mass Spectrometry Laboratory, Semel Institute of Neuroscience and Human Behavior, University of California, Los Angeles, California 90095.
authors.forename: Kym
authors.initials: K
authors.lastname: Faull
authors.affiliation: From the Ohio State Biochemistry Program, Department of Microbiology, The Ohio State University, Columbus, Ohio 43210 and ibba.1@osu.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Ibba
mesh_terms: D000215:Acylation; D000249:Adenosine Monophosphate / Q000235:genetics / Q000378:metabolism; D000409:Alanine / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D010652:Phenylalanine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D012331:RNA, Fungal; D012360:RNA, Transfer, Phe; D029701:Saccharomyces cerevisiae Proteins; D000249:Adenosine Monophosphate; D010649:Phenylalanine; D010652:Phenylalanine-tRNA Ligase; D000409:Alanine
keywords: Quality control; amino acid; aminoacyl tRNA synthetase; protein synthesis; transfer RNA (tRNA); translation