title: HemK, a class of protein methyl transferase with similarity to DNA methyl transferases, methylates polypeptide chain release factors, and hemK knockout induces defects in translational termination.
abstract: HemK, a universally conserved protein of unknown function, has high amino acid similarity with DNA-(adenine-N6) methyl transferases (MTases). A certain mutation in hemK gene rescues the photosensitive phenotype of a ferrochelatase-deficient (hemH) mutant in Escherichia coli. A hemK knockout strain of E. coli not only suffered severe growth defects, but also showed a global shift in gene expression to anaerobic respiration, as determined by microarray analysis, and this shift may lead to the abrogation of photosensitivity by reducing the oxidative stress. Suppressor mutations that abrogated the growth defects of the hemK knockout strain were isolated and shown to be caused by a threonine to alanine change at codon 246 of polypeptide chain release factor (RF) 2, indicating that hemK plays a role in translational termination. Consistent with such a role, the hemK knockout strain showed an enhanced rate of read-through of nonsense codons and induction of transfer-mRNA-mediated tagging of proteins within the cell. By analysis of the methylation of RF1 and RF2 in vivo and in vitro, we showed that HemK methylates RF1 and RF2 in vitro within the tryptic fragment containing the conserved GGQ motif, and that hemK is required for the methylation within the same fragment of, at least, RF1 in vivo. This is an example of a protein MTase containing the DNA MTase motif and also a protein-(glutamine-N5) MTase.
authors.affiliation: Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. knakahig@pobox.com
authors.forename: Kenji
authors.initials: K
authors.lastname: Nakahigashi
authors.forename: Naoko
authors.initials: N
authors.lastname: Kubo
authors.forename: Shin-ichiro
authors.initials: S
authors.lastname: Narita
authors.forename: Takeshi
authors.initials: T
authors.lastname: Shimaoka
authors.forename: Simon
authors.initials: S
authors.lastname: Goto
authors.forename: Taku
authors.initials: T
authors.lastname: Oshima
authors.forename: Hirotada
authors.initials: H
authors.lastname: Mori
authors.forename: Maki
authors.initials: M
authors.lastname: Maeda
authors.forename: Chieko
authors.initials: C
authors.lastname: Wada
authors.forename: Hachiro
authors.initials: H
authors.lastname: Inokuchi
mesh_terms: D000595:Amino Acid Sequence; D000693:Anaerobiosis; D001665:Binding Sites; D003001:Cloning, Molecular; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D008745:Methylation; D020125:Mutation, Missense; D018384:Oxidative Stress; D010101:Oxygen Consumption; D010443:Peptide Chain Termination, Translational / Q000235:genetics*; D011496:Protein Methyltransferases / Q000235:genetics* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013489:Suppression, Genetic; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D003470:Culture Media; D029968:Escherichia coli Proteins; D011496:Protein Methyltransferases; C092404:prmC protein, E coli; D001616:beta-Galactosidase