Chiral effects on the final step of an octahedron-shaped coordination capsule self-assembly.

The final step of the self-assembly of an octahedron-shaped coordination capsule was investigated by a novel theoretical method. Two different reaction pathways were detected and classified by the chiral types of constituents, which addressed the possibility of chiral effects on the self-assembly process.

Characterization of four extensin genes in Arabidopsis thaliana by differential gene expression under stress and non-stress conditions.

From Arabidopsis thaliana we isolated four different cDNAs that encode extensins, a family of cell-wall hydroxyproline-rich glycoproteins (HRGPs). Putative proteins (AtExt2-5) contained one open reading frame and characteristic Ser-(Pro)4 sequences organized in a high-order repetitive motif. AtExt2-5 genes were strongly expressed during rehydration after dehydration. They were also expressed after treatment with various amino acids. In particular, AtExt3 and five mRNAs were abundantly accumulated after treatment with L-Ser, Hyp, and L-Pro, which are major components of extensin proteins. The AtExt transcripts were strongly expressed in root tissues of both unbolted and bolted plants. The transcripts of AtExt2, 3, and 5 were also detected in the lower stem and flower buds, and that of AtExt4 was detected in bolted flowers. Therefore, we suggest that these four AtExt genes are novel extensin genes in A. thaliana, because the expression of atExt1, which has already been isolated from A. thaliana, was different from these.

Conservation of human and mouse basonuclins as a guide to important features of the protein.

The nucleotide sequence of mouse basonuclin has been determined from its cDNA by PCR and compared with the previously known sequence of human basonuclin. Overall, there is 88% identity in the encoded amino acid sequences, but some regions have been much more conserved than others. Zinc fingers 2 and 6, the region containing the nuclear localization signal and the region containing the serine stripe encode identical amino acid sequences in the two species, but differ by numerous silent nucleotide substitutions, suggesting that these regions are likely to be important for the functions of the protein common to the two species. Similarly, zinc fingers 1 and 5 diverge at only a single amino acid residue. In contrast, other regions of the sequence have diverged considerably, such as zinc fingers 3 and 4. The region adjacent to the N-terminus is very divergent and this aids in locating the translation start site. The highly conserved regions are likely to be essential for the common function of the proteins, and the more divergent regions may be either unconstrained or adapted to different requirements in the two species.

The deduced amino-acid sequence of the cloned cpxR gene suggests the protein is the cognate regulator for the membrane sensor, CpxA, in a two-component signal transduction system of Escherichia coli.

The cpxA gene of Escherichia coli K-12 encodes a membrane-associated sensor element of a two-component signal transduction system in bacteria. The cognate regulator element, however, has not yet been definitively identified. A 2.1-kb segment upstream from cpxA was amplified by polymerase chain reaction, cloned and sequenced. An open reading frame encoding 232 amino acids was found. It showed high homology to the regulator elements of two-component transduction systems. The newly identified gene, designated as cpxR, may encode the cognate protein receiving signals from CpxA.

Cellular and molecular physiology of Escherichia coli in the adaptation to aerobic environments.

Upon exposure to oxygen, Escherichia coli increases the expression of enzymes essential for aerobic respiration, such as components of the TCA cycle and terminal oxidase complexes. This increase requires the elimination of repression mediated by the Arc regulatory system under anaerobic conditions. Coordinately, the synthesis of enzymes that function in anaerobic processes such as fermentation decreases, partly due to the inactivation of the transcription factor Fnr. E. coli is thus able to adjust the levels of respiratory enzymes to fit its environmental circumstances, and in this case, reduces the production of the less energy efficient fermentation enzymes in favor of the aerobic pathways. In contrast to the advantage in energy production, aerobiosis brings a disadvantage to E. coli: the production of reactive oxygen species (ROS), i.e. superoxide anion radical (O2.-), hydrogen peroxide (H2O2), and hydroxyl radical (.OH). These byproducts of aerobic respiration damage many biological molecules, including DNA, proteins, and lipids. To alleviate the toxicity of these compounds, E. coli induces the synthesis of protective enzymes, such as Mn-dependent superoxide dismutase (SodA) and catalase I (HP I), and this induction is controlled by the regulatory proteins SoxRS, OxyR, and ArcAB. Thus, ArcAB, Fnr, SoxRS, and OxyR function in concert so that E. coli can optimize its energy production and growth rate. Fnr and SoxRS are cytoplasmic, DNA-binding proteins, and these regulatory systems utilize iron-sulfur clusters as cofactors which may directly sense the redox environment. OxyR is also a cytoplasmic, DNA-binding protein, and appears to respond to redox potential through the oxidation state of a specific cysteine residue. In the ArcAB system (which belongs to the family of two-component regulatory systems), ArcB, a membrane protein, functions as the sensor, and ArcA, a DNA-binding protein, directly controls target gene expression. Under anaerobic conditions, ArcB undergoes autophosphorylation and transphosphorylates ArcA, stimulating ArcA's DNA-binding activity. During aerobic growth, the transphosphorylation of ArcA does not occur. In this signal transduction mechanism, the ArcB C-terminal or "receiver" domain plays a critical role; that is, it stimulates or abolishes the transphosphorylation depending on the metabolic state of the cell, which in turn is influenced by the availability of oxygen. E. coli thus employs at least four global regulatory systems which monitor the cellular oxidative/metabolic conditions, and adjust the expression of more than 70 operons to give the organism a better aerobic life.

Phosphorylation/dephosphorylation of the receiver module at the conserved aspartate residue controls transphosphorylation activity of histidine kinase in sensor protein ArcB of Escherichia coli.

A membrane sensor protein, ArcB, recognizes anaerobic environments and signals the information to the cognate regulator, ArcA. This in vitro study presents the process and control of the signal-transduction phosphorylation. In the presence of ATP, the ArcB transmitter module undergoes autophosphorylation and then transfers the phosphoryl group to its own receiver module as well as to the ArcA receiver module. Results suggest that the phosphoryl group of the ArcB receiver module is released by an intrinsic phosphatase activity. D-Lactate inhibits the phosphatase activity that removes phosphoaspartate groups from the receiver module in ArcB, and the associated increase in phosphorylation of this module leads to an activation of transphosphorylation of subsequently added phosphohistidine groups on ArcB to the receiver module of ArcA. A similar effect was also observed in the presence of pyruvate, acetate, or NADH. Conversely, the non-phosphorylated ArcB receiver module completely inhibits the intermolecular transphosphorylation. Thus, the phosphorylation state of the ArcB receiver module controls signal transduction from ArcB to ArcA. Since the intrinsic phosphatase activity is inhibited by cellular metabolites that increasingly accumulate by anaerobiosis, the enzyme portion of ArcB may be involved in sensing anaerobic environments through cellular metabolites in vivo.

Three classes of C2H2 zinc finger proteins.

C2H2 zinc finger proteins probably comprise the largest family of regulatory proteins in mammals. Most zinc fingers bind to a cognate DNA. In addition to DNA, many of the proteins also bind to RNA or protein, and some bind to RNA only. The binding properties depend on the amino acid sequence of the finger domains and of the linker between fingers, as well as on the higher-order structures and the number of fingers. C2H2 zinc finger proteins contain from 1 to more than 30 figures. Based on the number and the pattern of the fingers, most of the proteins can be classified into one of three groups: triple-C2H2, multiple-adjacent-C2H2, and separated-paired-C2H2 finger proteins. In contrast to proteins with triple-C2H2 fingers, proteins with multiple-adjacent-C2H2 fingers can bind multiple, different ligands. Proteins with a number of separated-paired fingers bind to the target by means of only a single pair.

Nuclear localization of basonuclin in human keratinocytes and the role of phosphorylation.

Basonuclin is a zinc-finger protein found in basal cells of the epidermis. In human keratinocyte cultures, basonuclin is susceptible to serine-phosphorylation and the addition of the phosphatase inhibitor, okadaic acid, promotes accumulation of basonuclin in the cytoplasm. The region of basonuclin containing the nuclear localization signal of basonuclin is necessary for nuclear localization of the protein and Ser-541, located immediately C-terminal to the nuclear localization signal, is the principal phosphorylation site in vitro. A nearly complete basonuclin transiently expressed in cultured keratinocytes localizes predominantly in the nucleus, but substitution of aspartic acid for Ser-541 promotes cytoplasmic localization. The same substitution of Ser-537 has a similar but weaker effect. Substitution of both serine residues by alanine leads to nuclear localization. These results show that nuclear localization of basonuclin depends on serine dephosphorylation, primarily of Ser-541. Different subcellular locations of basonuclin in different keratinocyte subtypes are therefore most likely to be controlled by the state of phosphorylation of Ser-541.

Catabolite-like repression of extracellular enzyme production in Vibrio parahaemolyticus.

Production of extracellular amylase and protease in Vibrio parahaemolyticus was repressed by various carbohydrates present in the medium. In addition, the protease production was repressed very strongly by peptones or casamino acids. Cyclic adenosine 3', 5'-monophosphate (cyclic AMP) added exogenously could reverse the repression of amylase production, but not that of protease production irrespective of the "repressors" used. Mutants of V. parahaemolyticus, which resembled the reported cya (adenylate cyclase) and crp (cyclic AMP receptor protein) mutants of Escherichia coli and related organisms, were examined for the exoenzyme production. Amylase production in the mutants was defective, while their protease production was not defective, but rather accentuated as compared with that in the parental strain. These findings strongly suggest that amylase production is subject to catabolite repression mediated by cyclic AMP, whereas protease production is controlled by a repression mechanism which mimics in part, but may be distinct from catabolite repression.

Basonuclin, a zinc finger protein of keratinocytes and reproductive germ cells, binds to the rRNA gene promoter.

Basonuclin is a protein containing three pairs of C(2)H(2) zinc fingers. The protein has been found in the basal (germinal) cell layer of stratified squamous epithelia, such as the epidermis, and in germ cells of the testis and ovary. We show here that the human protein has specific affinity for a segment of the promoter of the gene for rRNA. Basonuclin interacts with two separate parts of the promoter, each possessing dyad symmetry. The upstream part, but not the downstream part, is known to bind UBF1, a transcription factor for rDNA. Basonuclin is likely to be a cell-type-specific regulatory protein for rDNA transcription.

arcA (dye), a global regulatory gene in Escherichia coli mediating repression of enzymes in aerobic pathways.

In Escherichia coli the levels of numerous enzymes associated with aerobic metabolism are decreased during anaerobic growth. In an arcA mutant the anaerobic levels of these enzymes are increased. The enzymes, which are encoded by different regulons, include members that belong to the tricarboxylic acid cycle, the glyoxylate shunt, the pathway for fatty acid degradation, several dehydrogenases of the flavoprotein class, and the cytochrome o oxidase complex. Transductional crosses placed the arcA gene near min O on the chromosomal map. Complementation tests showed that the arcA gene corresponded to the dye gene, which is also known as fexA, msp, seg, or sfrA because of various phenotypic properties [Bachmann, B. (1983) Microbiol. Rev. 47, 180-230]. A dye-deletion mutant was derepressed in the aerobic enzyme system. The term modulon is proposed to describe a set of regulons that are subject to a common transcriptional control.

Adaptation of Escherichia coli to redox environments by gene expression.

Escherichia coli is adroit in exploiting environmental energy sources to its greatest profit. A key strategy is to channel electron transport from donor to a terminal acceptor(s) so that the voltage drop is maximal. At the level of transcription, the goal is achieved by the interaction of three global regulatory systems, Fnr, NarL/NarX and ArcB/ArcA. In addition, the regulator FhlA is involved in a cascade-controlled pathway for the formate branch of the pyruvate fermentation pathway.

Purification and phosphorylation of the Arc regulatory components of Escherichia coli.

In Escherichia coli, a two-component signal transduction system, consisting of the transmembrane sensor protein ArcB and its cognate cytoplasmic regulatory protein ArcA, controls the expression of genes encoding enzymes involved in aerobic respiration. ArcB belongs to a subclass of sensors that have not only a conserved histidine-containing transmitter domain but also a conserved aspartate-containing receiver domain of the regulator family. 'ArcB (a genetically truncated ArcB missing the two transmembrane segments on the N-terminal end) and ArcA were purified from overproducing cells. Autophosphorylation of 'ArcB was revealed when the protein was incubated with [gamma-32P]ATP but not with [alpha-32P]ATP or [gamma-32P]GTP. When ArcA was incubated in the presence of 'ArcB and [gamma-32P]ATP, ArcA acquired radioactivity at the expense of the phosphorylated protein 'ArcB-32P. When a limited amount of 'ArcB was incubated with excess ArcA and [gamma-32P]ATP, ArcA-32P increased linearly with time. Under such conditions, for a given time period the amount of ArcA phosphorylated was proportional to the concentration of 'ArcB. Thus, 'ArcB acted as a kinase for ArcA. Chemical stabilities of the phosphorylated proteins suggested that 'ArcB-32P contained both a histidyl phosphate and an aspartyl phosphate(s) and that ArcA-32P contained only an aspartyl phosphate(s).

Mutational analysis of signal transduction by ArcB, a membrane sensor protein responsible for anaerobic repression of operons involved in the central aerobic pathways in Escherichia coli.

In Escherichia coli, the expression of a group of operons involved in aerobic metabolism is regulated by a two-component signal transduction system in which the arcB gene specifies the membrane sensor protein and the arcA gene specifies the cytoplasmic regulator protein. ArcB is a large protein belonging to a subclass of sensors that have both a transmitter domain (on the N-terminal side) and a receiver domain (on the C-terminal side). In this study, we explored the essential structural features of ArcB by using mutant analysis. The conserved His-292 in the transmitter domain is indispensable, indicating that this residue is the autophosphorylation site, as shown for other homologous sensor proteins. Compression of the range of respiratory control resulting from deletion of the receiver domain and the importance of the conserved Asp-533 and Asp-576 therein suggest that the domain has a kinetic regulatory role in ArcB. There is no evidence that the receiver domain enhances the specificity of signal transduction by ArcB. The defective phenotype of all arcB mutants was corrected by the presence of the wild-type gene. We also showed that the expression of the gene itself is not under respiratory regulation.