A conserved polar region in the cell division site determinant MinD is required for responding to MinE-induced oscillation but not for localization within coiled arrays

A region in the cell division site determinant MinD required for stimulation by MinE and which determines MinD topological specificity along coil-like structures has been identified. Structural modeling of dimeric MinD and sequence alignment of 24 MinD proteins revealed a conserved polar region in Gram-negative bacterial MinD proteins, corresponding to residues 92-94 of Neisseria gonorrhoeae MinD (MinD(Ng)). Using MinD(Ng) as a paradigm for MinD functionality in Gram-negative organisms, mutation of these conserved residues did not abrogate MinD(Ng) self-association, nor its interaction with MinE(Ng) and the cell division inhibitor MinC. Although the MinD(Ng) mutant dimerized in the presence of ATP, its ATPase activity was not stimulated by MinE(Ng), unlike wild-type MinD(Ng). GFP fusions to either MinD(Ng) or to Escherichia coli MinD bearing simultaneous or individual mutations to residues 92-94 localized within coiled arrays along the E. coli inner cell periphery, similar to wild-type GFP-MinD. However, unlike wild-type GFP-fusions, the mutant proteins were distributed uniformly throughout the array, despite the presence of MinE, which normally imparts topological specificity to MinD by inducing the latter to oscillate from pole-to-pole and away from midcell. Hence, despite localizing along the inner cell periphery as a polymeric structure, the mutant MinD proteins in this study have lost the ability to be efficiently stimulated by MinE(Ng), resulting in a loss of distinct pole-to-pole oscillation.     

Characterization of the MinD/ParA‐type ATPase FlhG in Vibrio alginolyticus and implications for function of its monomeric form

FlhG is a MinD/ParA‐type ATPase that works as a negative regulator for flagellar biogenesis. In Vibrio alginolyticus, FlhG functions antagonistically with the positive regulator FlhF to generate a single polar flagellum. Here, we examined the effects of ADP and ATP on the aggregation and dimerization of Vibrio FlhG. Purified FlhG aggregated after exposure to low NaCl conditions, and its aggregation was suppressed in the presence of ADP or ATP. FlhG mutants at putative ATP‐binding (K31A) or catalytic (D60A) residues showed similar aggregation profiles to the wild type, but ATP caused strong aggregation of the ATPase‐stimulated D171A mutant although ADP significantly suppressed the aggregation. Results of size exclusion chromatography of purified FlhG or Vibrio cell lysates suggested that FlhG exists as a monomer in solution, and ATP does not induce FlhG dimerization. The K31A and D60A mutants eluted at monomer fractions regardless of nucleotides, but ATP shifted the elution peak of the D171A mutant to slightly earlier, presumably because of a subtle conformational change. Our results suggest that monomeric FlhG can function in vivo, whose active conformation aggregates easily.     

ATPase/helicase motif mutants of Escherichia coli PriA protein essential for recombination-dependent DNA replication

BACKGROUNDS: PriA protein, a DEXH-type helicase with C2C2 zinc-finger motifs, plays essential roles in RecA-dependent modes of Escherichia coli chromosomal DNA replication, namely inducible and constitutive stable DNA replication (iSDR and cSDR respectively, which may be initiated from a D-loop or R-loop structure), and in repair of double-stranded DNA breaks generated by various genotoxic agents or spontaneously during the course of DNA replication. However, the roles of ATPase/DNA helicase activities in functions of PriA are not well understood. RESULTS: We have generated and characterized mutants of PriA protein carrying amino acid substitutions in its conserved ATPase/DNA helicase motifs, namely the Walker A, B and QXXGRXGR motifs. All these mutants were deficient in ATP hydrolysis and DNA helicase activities, but showed wild-type levels of D-loop DNA binding, except for the Walker B mutant which showed reduced DNA binding activity, suggesting that the helicase motifs are not directly involved in the DNA binding activity of PriA protein. They also rescued the low viability and UV-sensitivity of priA null cells. However, they did not rescue iSDR or cSDR-alternative modes of chromosomal DNA replication of the E. coli genome dependent on recombination functions-to the full extent. CONCLUSIONS: ATPase/DNA helicase activities of PriA protein are required for full-level DNA synthesis in recombination-dependent modes of DNA replication in E. coli.     

Expression of Neisseria gonorrhoeae cell division genes ftsZ, ftsE and minD is influenced by environmental conditions.

The activity of the promoter regions of the cell division genes ftsZ, ftsE, minC, minD and minE from Neisseria gonorrhoeae (Ng) was studied under different environmental conditions using lacZ translational fusions. The promoters of the minNg genes have not been previously determined and we identified promoter regions upstream of each gene (minCp, minDp and minEp). We determined that minDp had the strongest activity. Expression of the promoter regions of ftSZ(Ng) and ftsE(Ng), which we had previously identified, as well as minD(Ng), were then studied under conditions reflecting the environment of the genitourinary tract. These conditions included anaerobiosis, presence of isoleucine or urea (3 mM and 400 mM, respectively) and acidity of pH 6. Both beta-galactosidase expression and northern blot analysis indicated that all three genes were upregulated under anaerobiosis. The addition of isoleucine as well as media at pH 6 did not have any significant effects on the promoter activity of these genes while the presence of urea significantly decreased ftsZ(Ng) promoter activity. The expression of the minD(Ng) promoter region was analyzed during different growth phases and shown to follow the growth behavior of the culture. By contrast, the ftSZ(Ng) promoter activity continued to rise after the onset of the stationary phase. When gonococcal ftsZ promoter 1, (Pz1) was altered by site-directed mutagenesis, a significant decrease in the expression of ftsZ(Ng) was observed under both aerobic and anaerobic conditions. These data infer that gonococci regulate their cell division in response to different environments.     

Human Werner helicase interacting protein 1 (WRNIP1) functions as a novel modulator for DNA polymerase delta

Human WRNIP1, a Werner DNA helicase interacting protein 1, was expressed in insect cells and E. coli. The purified protein behaved as a homo-oligomeric complex with a native molecular mass indicative of an octamer, and the complex copurified with an ATPase activity that was stimulated by double-stranded DNA ends. As suggested by genetic studies of budding yeast WRNIP1/Mgs1, the purified human WRNIP1 complex interacted physically with human DNA polymerase delta (pol delta), stimulating its DNA synthesis activity more than fivefold in the presence or absence of proliferating cell nuclear antigen. Analysis of reaction products demonstrated the stimulation to be partly due to an increased processivity of pol delta but more importantly to an increase in its initiation frequency. Addition of ATP to reactions partially suppressed stimulation by WRNIP1. Furthermore, a mutant WRNIP1 lacking ATPase activity could stimulate pol delta normally but was insensitive to suppression by ATP. These results indicate that WRNIP1 functions as a modulator for initiation or restart events during pol delta-mediated DNA synthesis and that its ATPase activity is utilized to sense DNA ends and to regulate the extent of stimulation.     

Control of endotoxin activity and interleukin-1 production through regulation of lipopolysaccharide-lipoprotein binding by a macrophage factor.

Lipopolysaccharides (LPSs) extracted from gram-negative bacteria are much less active when bound to serum lipoproteins. We present evidence here that the binding of radiolabeled LPS extracted from Escherichia coli O113 and Salmonella typhimurium to lipoproteins in rabbit serum is increased 8 to 24 h after a single intravenous injection of homologous or heterologous LPS. Supernatants of activated macrophages containing interleukin-1 also stimulate increased binding. The isolated product of this binding does not induce the production of interleukin-1 by macrophages in vivo or in vitro and is unable itself to stimulate increased binding of LPS to lipoprotein. Normal rabbit sera spiked with lipoprotein fractions prepared from tolerant but not normal rabbit sera bind increased amounts of LPS. These data suggest that there may exist a self-regulated mechanism for decreasing the toxicity of LPS and the production of LPS-induced interleukin-1; this mechanism is controlled by a macrophage factor and functions through altering the binding of LPS to certain serum lipoproteins.     

The adherence of Escherichia coli mutants to mouse splenic lymphocytes

Strains of Escherichia coli that do not bind to lymphocytes can be converted to lymphocyte-binding strains by selecting for bacteriophage resistance. The mutants appear to fall into three classes: those whose binding can be inhibited by mannose, those whose binding can be inhibited by charged molecules, and those whose binding cannot be inhibited by either simple sugars or by charged molecules. The mannose-inhibitable mutants may bind via a bacterial lectin whereas the other mutants may bind via ionic interactions. Selection for phage resistance provides a simple way to obtain bacterial strains that bind to lymphocytes, which should facilitate the elucidation of the mechanism by which lymphocytes and bacteria interact.     

Role of V protein RNA binding in inhibition of measles virus minigenome replication

Measles virus V protein represses genome replication through a poorly understood mechanism, which led us to investigate whether V protein might be an RNA-binding modulatory factor. Recombinant V protein, expressed from transfected HEp-2 cells or E. coli, formed protein-RNA complexes with poly-guanosine (poly-G) or poly-U linked to agarose beads. RNA binding was not exclusive to ribonucleotide homopolymers as complex formation between V protein and an RNA molecule equivalent to the 3' terminal 107 bases of the measles virus genome was observed with an electrophoretic mobility shift assay (EMSA). The interaction with poly-G was used to further examine the RNA binding properties of V demonstrating that protein-RNA complex formation was dependent upon the unique Cys-rich carboxy terminus, a region also required to induce maximal repression of minireplicon-encoded reporter gene expression in transient assays. Surprisingly, two mutant proteins that contained Cys-to-Ala substitutions in the C-terminus were found to retain their ability to bind poly-G binding and repress minireplicon reporter gene expression indicating that neither activity was dependent on the integrity of all 7 C-terminal Cys residues. Additional genetic analysis revealed that amino acids 238-266 were necessary for efficient RNA binding and overlapped with residues (238-278) required for maximal repression induced by the C-terminal domain. In addition, a 10 amino acid deletion was identified (residues 238-247) that blocked RNA binding and repression indicating that these two activities were related.     

A Short Basic Domain Supports a Nucleic Acid-Binding Activity in the Rice Tungro Bacilliform Virus Open Reading Frame 2 Product

Little is known about the features of badnavirus open reading frame 2 products (P2). So far, no consensus functional domain has been found in these proteins. However, they all have in common at their C-terminus amino acids which may have the capacity to bind nucleic acids. Such capacity has already been established for cacao swollen shoot virus protein P2. We have looked for such a binding capacity of rice tungro bacilliform virus (RTBV) ORF 2 product. For this purpose, the protein was expressed as full-length or truncated versions inEscherichia coli.When used in nucleic acid-binding assays, complete RTBV P2 was shown to bind both DNA and RNA. This property may be related to a basic sequence, PPKKGIKRKYPA, localized at its C-terminus. Mutations were introduced into this sequence and revealed that four of the five basic residues, including a crucial lysine, are required for the binding to nucleic acids. Moreover, this sequence can confer binding capacity when it is fused to the N-terminus of nonbinding proteins.     

A comparative study of antimicrobial properties of crustinPm1 and crustinPm7 from the black tiger shrimp Penaeus monodon

Several isoforms of crustin have been identified in the black tiger shrimp Penaeus monodon. These cationic cysteine-rich antimicrobial peptides contain a single whey acidic protein (WAP) domain at the C-terminus and exhibit antimicrobial activity against both Gram-positive and Gram-negative bacteria. In this paper, we investigate the binding properties and antimicrobial actions of crustinPm1 and crustinPm7, the two most abundant crustin isoforms found in the haemocyte of P. monodon. Previously, crustinPm1 showed strong inhibition against Gram-positive bacteria, whilst crustinPm7 acted against both Gram-positive and Gram-negative bacteria. A binding study showed that both crustins can bind to Gram-positive and Gram-negative bacterial cells. Enzyme-linked immunosorbent (ELISA) assay suggested that crustins bind to the cell wall components, lipoteichoic acid (LTA) and lipopolysaccharide (LPS) with positive cooperativity of Hill slope (H) > 2. This indicates that at least two molecules of crustins interact with one LTA or LPS molecule. In addition, both crustins can induce bacterial agglutination and cause inner membrane permeabilization in Escherichia coli. Scanning Electron Microscopy (SEM) revealed the remarkable change on the cell surface of Staphylococcus aureus, Vibrio harveyi and E. coli after the bacteria were treated with the recombinant crustinPm7. Meanwhile, crustinPm1 can cause a visible change on the cell surface of S. aureus and E. coli only. This is in agreement with the fact that crustinPm1 has shown no antimicrobial activity against V. harveyi. It is likely that the antimicrobial activity of crustins mainly relies on their ability to agglutinate bacterial cells and to disrupt the physiochemical properties of bacterial surface.     

Gonococcal opacity: lectin-like interactions between Opa proteins and lipooligosaccharide.

Previous evidence from our laboratory suggested that the tight intercellular adhesions between the outer membranes of gonococci displaying the opacity colony phenotype occurred because Opa proteins expressed on one gonococcus adhered to the lipooligosaccharide (LOS) of the opposing bacterium (M.S. Blake, p. 51-66, in G. G. Jackson and H. Thomas, ed., The Pathogenesis of Bacterial Infections, 1985, and M. S. Blake and E. C. Gotschlich, p. 377-400, in M. Inouye, ed., Bacterial Outer Membranes as Model Systems, 1986). A noncompetitive inhibition assay used previously to determine the carbohydrate structures recognized by the major hepatic asialoglycoprotein receptor was modified to determine the gonococcal LOS structures that bind Opa proteins (R. T. Lee, Targeted Diagn. Ther. Ser. 4:65-84, 1991). The LOS carbohydrates used in these assays were LOS structures purified from pyocin LOS mutants of Neisseria gonorrhoeae 1291 described by K. C. Dudas and M. A. Apicella (Infect. Immun. 56:499-504, 1988) and further characterized by C. M. John et al. (J. Biol. Chem. 266:19303-19311, 1991). Purified gonococcal Opa proteins were incubated with each of the parent and mutant LOS, and the amount of binding of Opa proteins was measured by a direct enzyme-linked immunosorbent assay using the Opa-specific monoclonal antibody 4B12. The affinities of the Opa proteins for each of the LOS were determined indirectly by measuring the concentrations of Opa proteins that noncompetitively inhibited 50% of the binding of LOS-specific monoclonal antibodies. This concentration is inversely proportional to the affinity of the inhibitor (R. T. Lee, Targeted Diagn. Ther. Ser. 4:65-84, 1991). Our data suggest that the gonococcal Opa proteins tested had the highest affinity for the Gal beta 1-4GlcNAc residue present on the gonococcal lactoneoseries LOS. This affinity was comparable to that reported for the binding of the major hepatic asialoglycoprotein receptor to glycoconjugates containing terminal galactose and N-acetylgalactosamine (R. T. Lee, Targeted Diagn. Ther. Ser. 4:65-84, 1991). After sialylation of the lactoneoseries LOS, presumably on the terminal galactose residue, the interaction with the Opa proteins was ablated. Therefore, the gonococcal Opa-LOS and mammalian epithelial cell asialoglycoprotein receptor-carbohydrate interactions have quite similar specificities.     

Identification and cloning of a fur homolog from Neisseria gonorrhoeae.

The promoter region of the major iron-regulated protein of Neisseria gonorrhoeae, Fbp, has two regions that exhibit homology with the Escherichia coli consensus Fur-binding sequences. Gel retardation assays suggested that purified E. coli Fur bound to two sites within the Fbp promoter. The presence of a gonococcal Fur homolog was suggested by Southern hybridization under conditions of low stringency, which revealed a DNA locus that exhibited homology to the E. coli fur gene. Oligonucleotides derived from the conserved regions of fur genes of extremely diverse bacteria were used to amplify a 140-bp fragment of a putative gonococcal fur gene. This fragment was used to identify clones containing the entire gonococcal fur gene. After sequencing the gonococcal fur gene and its promoter region, we found that gonococcal Fur exhibited 50% identity with E. coli Fur at the amino acid level; however, it complemented two E. coli Fur- mutants. The presence of a Fur homolog in N. gonorrhoeae suggests that Fur-regulated genes are widely distributed among extremely diverse bacteria.     

Effects of Mutations within the SV40 Large T Antigen ATPase/p53 Binding Domain on Viral Replication and Transformation

The simian virus 40 (SV40) large T antigen is a 708 amino-acid protein possessing multiple biochemical activities that play distinct roles in productive infection or virus-induced cell transformation. The carboxy-terminal portion of T antigen includes a domain that carries the nucleotide binding and ATPase activities of the protein, as well as sequences required for T antigen to associate with the cellular tumor suppressor p53. Consequently this domain functions both in viral DNA replication and cellular transformation. We have generated a collection of SV40 mutants with amino-acid deletions, insertions or substitutions in specific domains of the protein. Here we report the properties of nine mutants with single or multiple substitutions between amino acids 402 and 430, a region thought to be important for both the p53 binding and ATPase functions. The mutants were examined for the ability to produce infectious progeny virions, replicate viral DNA in vivo, perform in trans complementation tests, and transform established cell lines. Two of the mutants exhibited a wild-type phenotype in all these tests. The remaining seven mutants were defective for plaque formation and viral DNA replication, but in each case these defects could be complemented by a wild-type T antigen supplied in trans. One of these replication-defective mutants efficiently transformed the REF52 and C3H10T1/2 cell lines as assessed by the dense-focus assay. The remaining six mutants were defective for transforming REF52 cells and transformed the C3H10T1/2 line with a reduced efficiency. The ability of mutant T antigen to transform REF52 cells correlated with their ability to induce increased levels of p53. This revised version was published online in August 2006 with corrections to the Cover Date.     

Analysis of functional domains on reovirus cell attachment protein sigma 1 using cloned S1 gene deletion mutants

Previously a reovirus (serotype 3) S1 gene cDNA was inserted into the lac cloning site of pUC13 and expressed in Escherichia coli to yield a sigma 1 fusion protein (F-sigma 1) capable of binding to mouse L fibroblasts and of agglutinating human red blood cells (S.A. Masri, L. Nagata, D. C. W. Mah, and P. W. K. Lee, 1986, Virology 149, 83-90). To probe the functional domains on the sigma 1 protein, restriction enzymes which divide the S1 gene into four segments (5'-I-II-III-IV-3') of similar size were used to generate five in-frame deletion mutants (D1-D5). Corresponding mutant forms of sigma 1 were expressed in E. coli and were assayed for (i) host cell (mouse L fibroblasts) binding activity; (ii) glycophorin (reovirus erythrocyte receptor) binding activity (R. W. Paul and P. W. K. Lee, 1987. Virology 159, 94-101 and (iii) recognizability by a library of neutralizing monoclonal anti-sigma 1 antibodies. It was found that mutant sigma 1 forms with segment III or segment IV deleted did not exhibit any detectable L-cell binding activity, whereas mutants with these two segments intact (but lacking segment II or segments I and II) were capable of attaching to L-cell receptors, albeit with reduced efficiencies. On the other hand, only F-sigma 1, but none of the mutants, could bind immobilized glycophorin. These data clearly suggest that the host cell binding domain of sigma 1 is distinct from its hemagglutination domain. Also, the five neutralizing anti-sigma 1 monoclonal antibodies tested were all found to recognize epitopes on either the middle segments or the carboxy-terminal half of sigma 1.     

Activation-induced cytidine deaminase: structure-function relationship as based on the study of mutants

Activation-induced cytidine deaminase (AID; gene symbol AICDA) is the key molecule required to induce immunoglobulin (Ig) class switch recombination (CSR) and somatic hypermutation (SHM) of the variable regions of Ig genes. Its deficiency causes a form of hyper-IgM (HIGM) syndrome. The study of natural AID mutants associated with HIGM as well as engineered mutants led to the characterization of the active domains of the protein. AID, through its cytidine deaminase activity, induces a targeted DNA lesion as an early step required for both CSR and SHM. Besides its cytidine deaminase activity, AID plays a further essential role in CSR, likely by recruiting CSR-specific cofactors by its C-terminus. A similar binding of SHM-specific cofactors to the N-terminal part is suggested by the functional characteristics of N(ter) AID artificial mutants. These data require confirmation in vivo. Finally, AID acts as a homo-, di-, or multimeric complex. Together, these data strongly suggest that AID, a master molecule for antibody diversification, exerts its activity on CSR not only as a cytidine deaminase enzyme but also as a docking protein, recruiting specific cofactors to a multimeric complex.     

A 101-kilodalton heme-binding protein associated with congo red binding and virulence of Shigella flexneri and enteroinvasive Escherichia coli strains.

The ability of Shigella flexneri to bind Congo red or hemin is associated with virulence. A 101-kilodalton (kDa) protein responsible for this phenotype (Crb+) in S. flexneri was identified by a tetramethylbenzidine staining procedure which detects heme-protein complexes in polyacrylamide gels. Labeling of cell-surface polypeptides with 125I revealed that the 101-kDa heme-binding protein is expressed on the cell surface. Expression of the protein was regulated by growth temperature and was found to be encoded by the large virulence plasmid of S. flexneri. Deletion mutants and a Tn5 insertion mutant which were negative for Congo red binding (Crb-) did not express the 101-kDa protein. Enteroinvasive Escherichia coli strains that were Crb+, and whose plasmids shared homology with the S. flexneri virulence plasmid, also expressed the 101-kDa protein. Expression of the protein in S. flexneri and enteroinvasive E. coli correlated with the presence of a 9.2-kilobase EcoRI fragment of these plasmids.     

C-myc标记肽对单链抗体与抗原结合活性的影响

目的 探讨在不同的免疫学检测方法中，与单链抗体相连的亲和标记肽对单链抗体与抗原结合活性的影响。方法 在制备“VH-连接链-VL”型结构抗乙酰胆碱受体单链抗体时，将c-myc标记肽与单链抗体的VL连接，应用先将抗c-myc抗体固定单链抗体的固相放射免疫测定法、直接将单链抗体与乙酰胆碱受体结合的液相放射免疫测定法，分别测定单链抗体与乙酰胆碱受体的结合活性。结果 在固相放射免疫测定法中，单链抗体不能与乙酰胆碱受体结合；在液相放射免疫测定法中，单链抗体则能与乙酰胆碱受体结合。结论 在应用不同的免疫学检测方法中，与单链抗体相连的c-myc标记肽可影响单链抗体与抗原的结合活性。     

Effect of insulin upon membrane-bound (Na+ + K+)-ATPase extracted from frog skeletal muscle.

1. Insulin stimulates the activity of membrane-bound ATPase isolated from frog skeletal muscle and from rat brain. The increase in activity of the membrane-bound ATPase system isolated from frog ranged from 9-8 to 53% at concentrations of Na+ (25 mM), K+ (10 mM), and ATP (2 mM) similar to those in in vivo experiments conducted previously (Moore, 1973). The increased activity of the membrane-bound ATPase is, therefore, at least as great as the insulin-induced increase in Na efflux (10-38%) from intact cells (Moore, 1973). If the concentration of Na+ is lowered to 4 mM and that of ATP lowered to 0-5 mM albumin, and 10(6) M, the increase in ouabain-inhibitable ATPase activity can reach as high as 400%. 2. Ouabain, at a concentration (10(-3) M) sufficient to inhibit stimulation of the frog ATPase by increasing Na from 4 to 25 mM, completely blocked the stimulation of ATPase activity due to insulin. 3. At 2 mM-ATP, 100 mM-Na+, and 20 mM-K+, conditions which maximally activate the (Na+ + K+)-ATPase, insulin did not increase the ATPase, activity. Stimulation was consistently seen at 10 mM-K+, 0-5 mM-ATP, and either 4 mM or 25 mM-Na+. 4. The finding that insulin does not stimulate the ATPase activity in conditions in which the (Na+ + K+)-ATPase component is maximally activated and especially the fact that ouabain can reproducibly inhibit insulin stimulation of the membrane-bound ATPase activity strongly suggest that interaction of insulin with its receptor upon the plasma membrane somehow stimulates the (Na+ + K+)-ATPase system (ouabain sensitive; ATP phosphohydrolase, EC (3.6.1.3). These results are consistent with previous studies of the effect of insulin upon Na efflux from intact cells (Moore, 1973) and support the previous conclusion that the component of Na efflux stimulated by insulin is active. The evidence suggests that insulin probably does not affect Vmax of the (Na+ + K+)-ATPase system, but may increase the affinity of the enzyme system to one or more effectors, most likely Na+, ATP, and perhaps K+. 5. Oxidized glutathione (2-7 X 10(-6) M), 10(-6) M, 10(-7) M, and 10(-8) M cyclic AMP did not affect the ATPase activity 10(-6)Malbumin, and . 6. The results are consistent with the view that the Na pump, (Na+ + K+)-ATPase, is intimately involved with the physiological action of insulin and may be transducer between the binding of insulin to its receptor on the plasma membrane and the cellular actions of insulin.     

Mammalian homologue of E. coli ras-like GTPase (ERA) is a possible apoptosis regulator with RNA binding activity

BACKGROUND: ERA (Escherichia coli Ras-like protein) is an E. coli GTP binding protein that is essential for proliferation. A DNA database search suggests that homologous sequences with ERA exist in various organisms including human, mouse, Drosophila, Caenorhabditis elegans and Antirrhinum majus. However, the physiological function of eukaryotic ERA-like proteins is not known. RESULTS: We have cloned cDNAs encoding the entire coding region of a human homologue (H-ERA) and a mouse homologue (M-ERA) of ERA. The mammalian homologue of ERA consists of a typical GTPase/GTP-binding domain and a putative K homology (KH) domain, which is known as an RNA binding domain. We performed transfection experiments with wild-type H-ERA or various H-ERA mutants. H-ERA possessing the amino acid substitution mutation into the GTPase domain induced apoptosis of HeLa cells, which was blocked by Bcl-2 expression. Deletion of the C-terminus, which contains a part of the KH domain, alleviated apoptosis by the H-ERA mutant, suggesting the importance of this domain in the function of H-ERA. We have also shown the RNA binding activity of H-ERA by pull-down experiments using RNA homopolymer immobilized on beads or recombinant H-ERA proteins. CONCLUSION: Our data suggest that H-ERA plays an important role in the regulation of apoptotic signalling with its GTPase/GTP binding domain.