[Proteome analysis using MALDI-TOF MS].
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Genome↗
Biomedical subjects
Chieko Wada
Publications and source records attributed to Chieko Wada.
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.
Amino Acid Sequence↗
Genome-wide analysis of deoxyadenosine methyltransferase-mediated control of gene expression in Escherichia coli.
Deoxyadenosine methyltransferase (Dam) methylates the deoxyadenine residues in 5'-GATC-3' sequences and is important in many cellular processes in Escherichia coli. We performed a computational analysis of the entire E. coli genome and confirmed that GATC sequences are distributed unevenly in regulatory regions, which suggests that Dam might regulate gene transcription. To test this, a high-density DNA microarray of 4097 E. coli genes was constructed and used to assess the gene expression profiles of the wild type and the dam-16::kam mutant strain grown under four different conditions. We also used two-dimensional electrophoretic analysis of the proteome to assess the protein profiles. The expression of a large number of genes was affected by the dam deficiency. Genes involved in aerobic respiration, stress and SOS responses, amino acid metabolism and nucleotide metabolism were expressed at higher levels in the mutant cells, especially in aerobic conditions. In contrast, transcription of genes participating in anaerobic respiration, flagella biosynthesis, chemotaxis and motility was decreased in the dam mutant strain under both aerobic and low aerobic conditions. Thus, Dam-controlled genes are involved in adjusting the metabolic and respiratory pathways and bacterial motility to suit particular environmental conditions. The promoters of most of these Dam-controlled genes were also found to contain GATC sequences that overlap with recognition sites for two global regulators, fumarate nitrate reduction (Fnr) and catabolite activator protein (CRP). We propose that Dam-mediated methylation plays an important role in the global regulation of genes, particularly those with Fnr and CRP binding sites.
Amino Acids↗
The ribosome modulation factor (RMF) binding site on the 100S ribosome of Escherichia coli.
During the stationary growth phase, Escherichia coli 70S ribosomes are converted to 100S ribosomes, and translational activity is lost. This conversion is caused by the binding of the ribosome modulation factor (RMF) to 70S ribosomes. In order to elucidate the mechanisms by which 100S ribosomes form and translational inactivation occurs, the shape of the 100S ribosome and the RMF ribosomal binding site were investigated by electron microscopy and protein-protein cross-linking, respectively. We show that (i) the 100S ribosome is formed by the dimerization of two 70S ribosomes mediated by face-to-face contacts between their constituent 30S subunits, and (ii) RMF binds near the ribosomal proteins S13, L13, and L2. The positions of these proteins indicate that the RMF binding site is near the peptidyl transferase center or the P site (peptidyl-tRNA binding site). These observations are consistent with the translational inactivation of the ribosome by RMF binding. After the "Recycling" stage, ribosomes can readily proceed to the "Initiation" stage during exponential growth, but during stationary phase, the majority of 70S ribosomes are stored as 100S ribosomes and are translationally inactive. We suggest that this conversion of 70S to 100S ribosomes represents a newly identified stage of the ribosomal cycle in stationary phase cells, and we have termed it the "Hibernation" stage.
Bacterial Proteins↗
Proteolytic activity of YibP protein in Escherichia coli.
Escherichia coli YibP protein (47.4 kDa) has a membrane-spanning signal at the N-terminal region, two long coiled-coil regions in the middle part, and a C-terminal globular domain, which involves amino acid sequences homologous to the peptidase M23/M37 family. A yibP disrupted mutant grows in rich medium at 37 degrees C but not at 42 degrees C. In the yibP null mutant, cell division and FtsZ ring formation are inhibited at 42 degrees C without SOS induction, resulting in filamentous cells with multiple nucleoids and finally in cell lysis. Five percent betaine suppresses the temperature sensitivity of the yibP disrupted mutation. The mutant has the same sensitivity to drugs, such as nalidixic acid, ethidium bromide, ethylmethane sulfonate, and sodium dodecyl sulfate, as the parental strain. YibP protein is recovered in the inner membrane and cytoplasmic fractions, but not in the outer membrane fraction. Results suggest that the coiled-coil regions and the C-terminal globular domain of YibP are localized in the cytoplasmic space, not in the periplasmic space. Purified YibP has a protease activity that split the substrate beta-casein.
Amino Acid Motifs↗