Cloning of a cDNA encoding a putative metal-transporting P-type ATPase from Arabidopsis thaliana.

Metal-transporting P-type ATPases were recently proposed to constitute a newly emerged sub-family of cation-transporting P-type ATPases, and are known to occur widely in prokaryotes and eukaryotes. However, no instance has been reported for higher plants. A cDNA clone encoding a metal-transporting P-type ATPase was thus searched for, if present, and was identified in Arabidopsis thaliana. The amino acid sequence, predicted from the determined nucleotide sequence for the cloned cDNA, shows all the critical features common to known metal-transporting P-type ATPases. This plant P-type ATPase has a typical metal-binding motif at its N-terminal portion. The newly isolated Arabidopsis gene, named PAA1, provides us with the first instance of putative metal-transporting P-type ATPases in higher plants. Some results of genomic analyses for this gene are also presented.

Clarification of the dimerization domain and its functional significance for the Escherichia coli nucleoid protein H-NS.

The Escherichia coli nucleoid protein, H-NS, functions as a global regulator for expression of a wide variety of genes. We recently analyzed the structure-function relationship of H-NS with special reference to the domains responsible for transcriptional repression and DNA-binding, respectively. However, identification of the presumed dimerization domain of H-NS and its functional significance was elusive. To address this particular issue, we first examined a set of N-terminally or C-terminally truncated forms of H-NS, in terms of their so-called dominant-negative effect on the in vivo function of the wild-type H-NS. The results showed that certain truncated forms exhibit such a dominant-negative effect, but others did not. As judged by the results of the dominant-negative effect, it was assumed that a relatively central portion of H-NS extending from residues 21 to 63 is involved in dimerization. This was confirmed by an in vitro chemical cross-linking analysis and a gel filtration analysis with these truncated forms of H-NS. Furthermore, the use of the dominant-negative phenotype, caused by a truncated form of H-NS (named N91), allowed us to isolate a missense mutant, which was expected to be specifically defective in dimerization. This mutant had an amino acid substitution at position 30 (Leu30 to Pro) in N91 consisting of the N-terminal 91 amino acids of H-NS. This mutant was indeed defective in the in vitro ability to form a heterodimer with the wild-type H-NS. When this particular single amino acid substitution was introduced into the full-length H-NS, the resultant H-NS mutant had lost the ability to form dimers in vitro and to function as a transcriptional repressor. These findings collectively provided us with evidence that the ability of H-NS to form a dimer is crucial for H-NS to function as a transcriptional repressor.

Systematic mutational analysis revealing the functional domain organization of Escherichia coli nucleoid protein H-NS.

The Escherichia coli H-NS protein is one of the major constituents of the nucleoid structure. This protein has been implicated not only in the compact organization of the nucleoid structure, but also in the global regulation of gene expression. H-NS negatively regulates the transcription of a number of apparently unlinked genes on the chromosome, suggesting that it functions as a global transcriptional repressor. In this study, on systematic mutational analysis of hns, three distinct functional domains were found in H-NS, which appear to be responsible for DNA-binding, transcriptional repression and protein-protein interaction (dimerization and/or oligomerization), respectively. We first isolated a number of hns mutations which resulted in derepression of the proVWX operon. These included 20 independent missence mutations each resulting in a single amino acid substitution, and six nonsense mutations each giving a C-terminally truncated form of H-NS. The substituted amino acids were revealed to be located non-randomly in the primary sequence of H-NS. This set of hns mutants was examined extensively in terms of phenotypes and biochemical properties. Based on the in vivo and in vitro results, together with the locations of the altered amino acids, three distinct functional domains were identified in H-NS. Mutations in the C-terminal domain resulted in a loss of its DNA-binding ability, suggesting that this domain is directly involved in its binding to DNA. The N-terminal domain was suggested to be involved in the ability to repress transcription. Mutations in this region abolished its ability to repress the transcription of proV, in vivo and in vitro, without loss of its DNA-binding activity. None of the mutants examined was impaired in the formation of a dimer and/or oligomers, suggesting that the central region of H-NS is involved in oligomerization. These results are discussed with special reference to the molecular mechanism underlying the function of H-NS as a transcriptional repressor. In addition, expression of the bgl operon was found to be affected by only a subset of hns mutations in a highly allele-specific manner. This finding is also addressed with regard to a unique regulatory mechanism (i.e. silencing) for the bgl operon, which is partly mediated by H-NS.

Sequence of a fission yeast gene encoding a protein with extensive homology to eukaryotic elongation factor-1 gamma.

A polypeptide with an apparent molecular mass of 23 kDa was identified, that exhibited an affinity to a 491-bp DNA derived from one of the Schizosaccharomyces pombe centromeric DNAs (cen1). After determining its N-terminal amino acid (aa) sequence, a Sz. pombe genomic DNA encompassing the coding sequence of the isolated protein was cloned, and a 2.3-kb genomic DNA region sequenced. Further sequence analysis of cDNA clones, originating from this particular genomic region, confirmed the existence of an open reading frame with a short intron, which encodes a 409-aa protein with striking homology to eukaryotic elongation factor-1 gamma.

An Arabidopsis protein that interacts with the cytokinin-inducible response regulator, ARR4, implicated in the His-Asp phosphorylay signal transduction.

Previously, Arabidopsis thaliana was shown to possess a set of response regulators (ARR-series), which are implicated in the prokaryotic type of signal transduction mechanism, generally referred to as the His-Asp phosphorylay. Among them, ARR4 is a typical phospho-accepting response regulator, whose expression was recently demonstrated to be rapidly induced by a cytokinin-treatment of the plant. To gain insight into the presumed His-Asp phosphotransfer signaling mechanism as well as the role of ARR4 in this higher plant, in this study we adopt the widely used yeast two-hybrid system, and report the identification of an Arabidopsis protein that has an ability to interact physically with the cytokinin-inducible ARR4 response regulator.

Identification of the DNA binding surface of H-NS protein from Escherichia coli by heteronuclear NMR spectroscopy.

The DNA binding domain of H-NS protein was studied with various N-terminal deletion mutant proteins and identified by gel retardation assay and heteronuclear 2D- and 3D-NMR spectroscopies. It was shown from gel retardation assay that DNA binding affinity of the mutant proteins relative to that of native H-NS falls in the range from 1/6 to 1/25 for H-NS(60-137), H-NS(70-137) and H-NS(80-137), whereas it was much weaker for H-NS(91-137). Thus, the DNA binding domain was defined to be the region from residue A80 to the C-terminus. Sequential nuclear Overhauser effect (NOE) connectivities and those of medium ranges revealed that the region of residues Q60-R93 in mutant protein H-NS(60-137) forms a long stretch of disordered, flexible chain, and also showed that the structure of the C-terminal region (residues A95-Q137) in mutant H-NS(60-137) was nearly identical to that of H-NS(91-137). 1H and 15N chemical shift perturbations induced by complex formation of H-NS(60-137) with an oligonucleotide duplex 14-mer demonstrated that two loop regions, i.e. residues A80-K96 and T110-A117, play an essential role in DNA binding.

Expression of Arabidopsis response regulator homologs is induced by cytokinins and nitrate.

We examined cytokinin and nitrate responsiveness in gene expression of five distinct response regulator homologs (ARR3-ARR7) in the leaves of nitrogen-starved Arabidopsis plants. The transcripts accumulated after spraying the shoots with t-zeatin. The induction of accumulation was highly specific for cytokinins. The transcripts also accumulated by supply of nitrate to the culture medium. These findings suggest that ARRs are involved in inorganic nitrogen signal transduction mediated by cytokinin as in the case of ZmCip1, a response regulator homolog recently identified in maize.

Solution structure of the DNA binding domain of a nucleoid-associated protein, H-NS, from Escherichia coli.

The three-dimensional structure of the C-terminal domain (47 residues) obtained from the hydrolysis of H-NS protein with bovine trypsin was determined by NMR measurements and distance geometry calculations. It is composed of an antiparallel beta-sheet, an alpha-helix and a 3(10)-helix which form a hydrophobic core, stabilizing the whole structure. This domain has been found to bind to DNA. Possible DNA binding sites are discussed on the basis of the solution structure of the C-terminal domain of H-NS.

Yeast gene which suppresses the defect in protein export of a secY mutant of E. coli.

To find factors participating in protein translocation in yeast, we screened a yeast genomic library for genes which, when introduced into Escherichia coli, suppressed secY24, a temperature sensitive mutation of an essential integral membrane protein (SecY) required for protein export. We isolated and characterized a gene (YSY6) which improved the translocation of the OmpA protein in mutant strain IQ85(secY24). It could also suppress another mutant [rplO215(Am)], in which the level of expression of the SecY protein is decreased at high-temperature. The YSY6 gene encodes a small amphiphilic peptide consisting of 65 amino acids, which can be expressed in E. coli cells.

The rpoD gene functions as a multicopy suppressor for mutations in the chaperones, CbpA, DnaJ and DnaK, in Escherichia coli.

The CbpA protein is an analog of the DnaJ molecular chaperone of Escherichia coli. The dnaJ- cbpA- double-null mutant exhibits severe defects in cell growth, namely, a very narrow temperature range for growth. To gain insight into the functions of CbpA as well as DnaJ, we isolated a multicopy suppressor gene that permits this dnaJ- cbpA- mutant to grow normally at low temperatures. The suppressor gene was identified as rpoD, the gene that encodes the major sigma 70. The biological implications of this finding are examined and discussed.

The Escherichia coli nucleoid protein H-NS functions directly as a transcriptional repressor.

The H-NS protein is a major constituent of the Escherichia coli nucleoid structure and is implicated in the compact organization of the chromosome. Based on recent genetic evidence, this protein appears to influence the transcription of a variety of apparently unlinked genes on the chromosome, although the underlying molecular mechanism is not fully understood. In this study, we carried out a series of in vitro transcription assays including purified H-NS with special reference to the osmotically inducible proV promoter of the proVWX operon (or proU), whose expression is known to be derepressed by lesions of the hns (osmZ) gene. Here, H-NS was revealed to selectively inhibit an early step(s) of proV transcription initiation through its direct binding to the promoter region. It was thus demonstrated that H-NS functions directly as a transcriptional repressor. Under the in vitro conditions used, this in vitro inhibitory effect of H-NS was affected by changes in the superhelical density of template DNAs and more significantly by the concentration of potassium (K+) ions. These results are also discussed with regard to the mechanism underlying regulation of the proV promoter in response to the medium osmolarity.

Multicopy suppression: an approach to understanding intracellular functioning of the protein export system.

Escherichia coli genes were cloned onto a multicopy plasmid and selected by the ability to restore growth and protein export defects caused by a temperature-sensitive secY or secA mutation. When secA51 was used as the primary mutation, only clones carrying groE, which specifies the chaperonin class of heat shock protein, were obtained. Selection using secY24 yielded three major classes of genes. The first class encodes another heat shock protein, HtpG; the most frequently obtained second class encodes a neutral histonelike protein, H-NS; and the third class, msyB, encodes a 124-residue protein of which 38 residues are acidic amino acids. Possible mechanisms of suppression as well as the significance and limitations of the multicopy suppression approach are discussed.

Escherichia coli sec mutants accumulate a processed immature form of maltose-binding protein (MBP), a late-phase intermediate in MBP export.

Protein translocation across the Escherichia coli cytoplasmic membrane may consist of several temporally or topographically distinct steps. Although early events in the translocation pathway have been characterized to some extent, the mechanisms responsible for the trans-bilayer movement of a polypeptide are only poorly understood. This article reports on our attempts to dissect the translocation pathway in vivo. A processed form of maltose-binding protein (MBP) was detected in the spheroplasts of secY and secA temperature-sensitive mutant cells that had been pulse-labeled at the permissive temperature (30 degrees C). This species of molecule was found to have an electrophoretic mobility identical to that of the mature MBP, but a considerable fraction of it was inaccessible to externally added protease. It had not attained the protease-resistant conformation characteristically observed for the exported mature protein. The radioactivity associated with this species decreased during chase and was presumably converted into the exported mature form, a process that required energy, probably the proton motive force, as demonstrated by its inhibition by an energy uncoupler. The spheroplast-associated processed form was more predominantly observed in the presence of a low concentration of chloramphenicol. A similar intermediate was also detected for beta-lactamase in wild-type cells. These results suggest that in a late phase of translocation, the bulk of the polypeptide chain can move through the membrane in the absence of the covalently attached leader peptide, and the secA-secY gene products are somehow involved in this process. We termed the processed intermediates processed immature forms.

Importance of stereospecific positioning of the upstream cis-acting DNA element containing a curved DNA structure for the functioning of the Escherichia coli proV promoter.

The mechanism by which the Escherichia coli proV promoter is activated more than 100-fold in response to the medium osmolarity, without the help of any known trans-acting activators, is not yet fully understood. In this context, it has recently begun to be realized that structural features, not the primary sequences, of cis-acting DNA elements may be important for transcriptional regulation in prokaryotes. From this point of view, in this study the proV promoter was characterized by constructing a series of spacer-insertion mutants in a proV-lacZ fusion on the chromosome. Here it was found that the upstream cis-acting sequence must be positioned stereospecifically with respect to the principal -35 and -10 regions for the proV promoter to be fully activated. In this regard, it was suggested that an overall DNA structure, particularly DNA curvature, is an important cis-acting parameter for activation of the proV promoter.

Quantitative control of the stationary phase-specific sigma factor, sigma S, in Escherichia coli: involvement of the nucleoid protein H-NS.

In Escherichia coli, recent intensive studies revealed that expression of a certain subset of genes is under the control of the stationary phase-specific sigma factor, sigma S, which is encoded by the rpoS gene. Since sigma S functions predominantly under certain growth conditions, its activity and/or cellular content has accordingly to be tightly controlled, however, the underlying molecular mechanism is at present unclear. We previously demonstrated that expression of the cbpA gene, encoding an analogue of the DnaJ molecular chaperone, is largely dependent on sigma S function. Here we have found that a mutational lesion of the hns gene, which encodes one of the well-characterized nucleoid proteins, H-NS, affects the cellular content of sigma S remarkably and consequently affects the expression of cbpA. Enhanced accumulation of sigma S in hns deletion cells was particularly observed in the logarithmic growth phase and was demonstrated to result from an elevated translational efficiency of rpoS mRNA and also from an increased stability of newly synthesized sigma S. Although H-NS is known to influence the transcription of a number of apparently unlinked genes on the chromosome, in this study we provide a novel instance in which H-NS is deeply implicated in post-transcriptional regulation(s) of the expression of rpoS. As to physiological relevance, it was also demonstrated that hns deletion cells exhibit an extreme thermotolerance even in the logarithmic growth phase, presumably because of the enhanced accumulation of sigma S.

Autoregulatory expression of the Escherichia coli hns gene encoding a nucleoid protein: H-NS functions as a repressor of its own transcription.

The Escherichia coli nucleoid protein, H-NS (or H1a), appears to influence the regulation of a variety of unrelated E. coli genes and operons. To gain an insight into the regulation of the hns gene itself, we constructed in this study a hns-lacZ transcriptional fusion gene and inserted a single copy at the att lambda locus on the E. coli chromosome. Expression of hns transcription appeared to be moderately regulated in a growth phase-dependent manner. It also emerged that hns transcription is under negative autoregulation, at least in the logarithmic growth phase. The results of in vitro transcription experiments confirmed that H-NS functions as a repressor for its own transcription. Thus, H-NS was shown to exhibit relatively high affinity for the DNA sequence encompassing the hns promoter region, as compared with a non-specific sequence. These results support the view that the nucleoid protein, H-NS, can function as a transcriptional regulator.

rpoS function is essential for bgl silencing caused by C-terminally truncated H-NS in Escherichia coli.

From evolutionary and physiological viewpoints, the Escherichia coli bgl operon is intriguing because its expression is silent (Bgl(-) phenotype), at least under several laboratory conditions. H-NS, a nucleoid protein, is known as a DNA-binding protein involved in bgl silencing. However, we previously found that bgl expression is still silent in a certain subset of hns mutations, each of which results in a defect in its DNA-binding ability. Based on this fact, we proposed a model in which a postulated DNA-binding protein(s) has an adapter function by interacting with both the cis-acting element of the bgl promoter and the mutated H-NS. To identify such a presumed adapter molecule, we attempted to isolate mutants exhibiting the Bgl(+) phenotype in the background of hns60, encoding the mutant H-NS protein lacking the DNA-binding domain by random insertion mutagenesis with the mini-Tn10cam transposon. These isolated mutations were mapped to five loci on the chromosome. Among these loci, three appeared to be leuO, hns, and bglJ, which were previously characterized, while the other two were novel. Genetic analysis revealed that the two insertions are within the rpoS gene and in front of the lrhA gene, respectively. The former encodes the stationary-phase-specific sigma factor, sigma(S), and the latter encodes a LysR-like DNA-binding protein. It was found that sigma(S) is defective in both types of mutant cells. These results showed that the rpoS function is involved in the mechanism underlying bgl silencing, at least in the hns60 background used in this study. We also examined whether the H-NS homolog StpA has such an adapter function, as was previously proposed. Our results did not support the idea that StpA has an adapter function in the genetic background used.