ArfA recruits release factor 2 to rescue stalled ribosomes by peptidyl-tRNA hydrolysis in Escherichia coli.

The ribosomes stalled at the end of non-stop mRNAs must be rescued for productive cycles of cellular protein synthesis. Escherichia coli possesses at least three independent mechanisms that resolve non-productive translation complexes (NTCs). While tmRNA (SsrA) mediates trans-translation to terminate translation, ArfA (YhdL) and ArfB (YaeJ) induce hydrolysis of ribosome-tethered peptidyl-tRNAs.

Functional and expressional analyses of the anti-FlhD4C2 factor gene ydiV in Escherichia coli.

Although Escherichia coli and Salmonella enterica serovar Typhimurium have a similar flagellar regulatory system, the response of flagellar synthesis to nutrient conditions is quite different between the two: that is, in low-nutrient conditions, flagellar synthesis is inhibited in Salmonella and enhanced in E. coli. In Salmonella, this inhibition is mediated by an anti-FlhD(4)C(2) factor, YdiV, which is expressed in low-nutrient conditions and binds to FlhD(4)C(2) to inhibit the expression of the class 2 flagellar genes.

trans-translation-mediated tight regulation of the expression of the alternative ribosome-rescue factor ArfA in Escherichia coli.

Ribosomes translating mRNA without an in-frame stop codon (non-stop mRNA) stall at its 3' end. In eubacteria, such ribosomes are rescued by SsrA-mediated trans-translation. Recently, we have shown that Escherichia coli ArfA (formerly YhdL) also rescues stalled ribosomes by a mechanism distinct from that of trans-translation.

The transcript from the σ(28)-dependent promoter is translationally inert in the expression of the σ(28)-encoding gene fliA in the fliAZ operon of Salmonella enterica serovar Typhimurium.

There are three classes of promoters for flagellar operons in Salmonella. Class 2 promoters are transcribed by σ(70) RNA polymerase in the presence of an essential activator, FlhD(4)C(2), and activated by an auxiliary regulator, FliZ. Class 3 promoters are transcribed by σ(28) RNA polymerase and repressed by an anti-σ(28) factor, FlgM.

FliZ acts as a repressor of the ydiV gene, which encodes an anti-FlhD4C2 factor of the flagellar regulon in Salmonella enterica serovar typhimurium.

YdiV acts as an anti-FlhD4C2 factor, which negatively regulates the class 2 flagellar operons in poor medium in Salmonella enterica serovar Typhimurium. On the other hand, one of the class 2 flagellar genes, fliZ, encodes a positive regulator of the class 2 operons. In this study, we found that the FliZ-dependent activation of class 2 operon expression was more profound in poor medium than in rich medium and not observed in the ydiV mutant background.

Escherichia coli YaeJ protein mediates a novel ribosome-rescue pathway distinct from SsrA- and ArfA-mediated pathways.

Accumulation of stalled ribosomes at the 3' end of mRNA without a stop codon (non-stop mRNA) is supposed to be toxic to bacterial cells. Escherichia coli has at least two distinct systems to rescue such stalled ribosomes: SsrA-dependent trans-translation and ArfA-dependent ribosome rescue. Combination of the ssrA and arfA mutations is synthetically lethal, suggesting the significance of ribosome rescue.

EAL domain protein YdiV acts as an anti-FlhD4C2 factor responsible for nutritional control of the flagellar regulon in Salmonella enterica Serovar Typhimurium.

Flagellar operons are divided into three classes with respect to their transcriptional hierarchy in Salmonella enterica serovar Typhimurium. The class 1 gene products FlhD and FlhC act together in an FlhD(4)C(2) heterohexamer, which binds upstream of the class 2 promoters to facilitate binding of RNA polymerase. In this study, we showed that flagellar expression was much reduced in the cells grown in poor medium compared to those grown in rich medium.

Measurement of sialic acid content is insufficient to assess bioactivity of recombinant human erythropoietin.

Assessment of biological potency and its comparison with clinical effects are important in the quality control of therapeutic glycoproteins. Animal models are usually used for evaluating bioactivity of these compounds. However, alternative methods are required to simplify the bioassay and avoid ethical issues associated with animal studies.

Suppression by enhanced RpoE activity of the temperature-sensitive phenotype of a degP ssrA double mutant in Escherichia coli.

SsrA is a small RNA playing a crucial role in trans-translation, which leads to rescue of stalled ribosomes on or at the end of mRNA and addition of the degradation tag to a growing polypeptide. The lack of SsrA has been shown to enhance the temperature-sensitive (ts) phenotype of an E. coli strain defective in the degP gene, which encodes one of the periplasmic proteases.

Production of novel anti-recombinant human erythropoietin monoclonal antibodies and development of a sensitive enzyme-linked immunosorbent assay for detection of bioactive human erythropoietin.

Erythropoietin (EPO) is a growth factor, regulating the proliferation and differentiation of erythroid progenitor cells. In this study, we generated five monoclonal antibodies (mAbs) that reacted specifically with recombinant human EPO (rhEPO). Epitope exclusion and other experiments showed that the mAbs obtained were divided into two groups, differing in recognition sites for rhEPO: group 1 mAbs recognize the N-terminal region of rhEPO, whereas group 2 mAbs seem to recognize a conformation-dependent epitope.

Expressed and cryptic flagellin genes in the H44 and H55 type strains of Escherichia coli.

Bacterial H antigens are specified by flagellin molecules, which constitute the flagellar filament. Escherichia coli 781-55 and E2987-73 are the type strains for H44 and H55 antigens, respectively. Unlike E.

Plasticity of the domain structure in FlgJ, a bacterial protein involved in flagellar rod formation.

Bacterial flagellar rod structure is built across the peptidoglycan (PG) layer. A Salmonella enterica flagellar protein FlgJ is believed to consist of two functional domains, the N-terminal half acting as a scaffold or cap essential for rod assembly and the C-terminal half acting as a PG hydrolase (PGase) that makes a hole in the PG layer to facilitate rod penetration. In this study, molecular data analyses were conducted on FlgJ data sets sampled from a variety of bacterial species, and three types of FlgJ homologs were identified: (i) "canonical dual-domain" type found in beta- and gamma-proteobacteria that has a domain for one of the PGases, acetylmuramidase (Acm), at the C terminus, (ii) "non-canonical dual-domain" type found in the genus Desulfovibrio (delta-proteobacteria) that bears a domain for another PGase, M23/M37-family peptidase (Pep), at the C terminus and (iii) "single-domain" type found in phylogenetically diverged lineages that lacks the Acm or Pep domain.

FliT acts as an anti-FlhD2C2 factor in the transcriptional control of the flagellar regulon in Salmonella enterica serovar typhimurium.

Flagellar operons are divided into three classes with respect to their transcriptional hierarchy in Salmonella enterica serovar Typhimurium. The class 1 gene products FlhD and FlhC act together in an FlhD(2)C(2) heterotetramer, which binds upstream of the class 2 promoters to facilitate binding of RNA polymerase. Class 2 expression is known to be enhanced by a disruption mutation in a flagellar gene, fliT.

FljA-mediated posttranscriptional control of phase 1 flagellin expression in flagellar phase variation of Salmonella enterica serovar Typhimurium.

Flagellar phase variation of Salmonella is a phenomenon where two flagellin genes, fliC (phase 1) and fljB (phase 2), are expressed alternately. This is controlled by the inversion of a DNA segment containing the promoter for the fljB gene. The fljB gene constitutes an operon with the fljA gene, which encodes a negative regulator for fliC expression.

Two DNA invertases contribute to flagellar phase variation in Salmonella enterica serovar Typhimurium strain LT2.

Salmonella enterica serovar Typhimurium strain LT2 possesses two nonallelic structural genes, fliC and fljB, for flagellin, the component protein of flagellar filaments. Flagellar phase variation occurs by alternative expression of these two genes. This is controlled by the inversion of a DNA segment, called the H segment, containing the fljB promoter.

Interactions between bacterial flagellar axial proteins in their monomeric state in solution.

The axial structure of the bacterial flagellum is composed of many different proteins, such as hook protein and flagellin, and each protein forms a short or long axial segment one after another in a well-defined order along the axis. Under physiological conditions, most of these proteins are stable in the monomeric state in solution, and spontaneous polymerization appears to be suppressed, as demonstrated clearly for flagellin, probably to avoid undesirable self-assembly in the cytoplasmic space. However, no systematic studies of the possible associations between monomeric axial proteins in solution have been carried out.

The ClpXP ATP-dependent protease regulates flagellum synthesis in Salmonella enterica serovar typhimurium.

The ClpXP protease is a member of the ATP-dependent protease family and plays a dynamic role in the control of availability of regulatory proteins and the breakdown of abnormal and misfolded proteins. The proteolytic activity is rendered by the ClpP component, while the substrate specificity is determined by the ClpX component that has ATPase activity. We describe here a new role of the ClpXP protease in Salmonella enterica serovar Typhimurium in which ClpXP is involved in the regulation of flagellum synthesis.

The Salmonella FlgA protein, a putativeve periplasmic chaperone essential for flagellar P ring formation.

P ring is a periplasmic substructure of the flagellar basal body and is believed to connect with the peptidoglycan layer in Salmonella. Two flagellar genes, flgA and flgI, are known to be indispensable for P ring formation. The flgI gene encodes the component protein of the P ring.

Structure and expression of the fliA operon of Salmonella typhimurium.

The fliA gene encodes the flagellum-specific sigma factor sigma28 In Salmonella typhimurium. The transcription in vivo and in vitro of this gene was analysed and it was found that there are two promoters for the expression of this gene. One is a class 2 promoter which is recognized by sigma70-RNA polymerase in the presence of the FlhD and FlhC activator proteins.