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.
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.
Nakahigashi
Kenji
K
Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. knakahig@pobox.com
Kubo
Naoko
N
Narita
Shin-ichiro
S
Shimaoka
Takeshi
T
Goto
Simon
S
Oshima
Taku
T
Mori
Hirotada
H
Maeda
Maki
M
Wada
Chieko
C
Inokuchi
Hachiro
H
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
D003470:Culture Media; D029968:Escherichia coli Proteins; D011496:Protein Methyltransferases; C092404:prmC protein, E coli; D001616:beta-Galactosidase