Biomedical subjects

Akira Ishihama

Publications and source records attributed to Akira Ishihama.

At least 19 recordsLinked to original sources

PubMed2003

Rpb7 subunit of RNA polymerase II interacts with an RNA-binding protein involved in processing of transcripts.

Rpb4-Rpb7, a dissociable subcomplex of RNA polymerase II (pol II), is required for transcription initiation. To understand the role of Rpb7 in transcription initiation or other processes in transcription, we carried out a two-hybrid screen for proteins that interact with Rpb7 of the fission yeast Schizosaccharomyces pombe. The screen identified the S.pombe homolog of the Saccharomyces cerevisiae Nrd1, an RNA-binding protein implicated in 3' end formation of small nucleolar and small nuclear RNAs transcribed by pol II. The S.pombe protein, named Seb1 for seven binding, was essential for cell viability, and bound directly to Rpb7 in vitro. Saccharomyces cerevisiae Rpb7 also interacted with Nrd1, indicating that the interaction is conserved in evolution. Glu166 and/or Asp167 of S.pombe Rpb7, residues near the C-terminus of the 172 amino acid protein, were found to be important for its interaction with Seb1. Our results suggest that Rpb7 may function to anchor a processing factor to the pol II apparatus, thereby coupling RNA processing to transcription. The role for Rpb7 is consistent with its location in the pol II complex determined by recent structural studies.

Amino Acid Sequence↗

PubMed2003

Recruitment of sigma54-RNA polymerase to the Pu promoter of Pseudomonas putida through integration host factor-mediated positioning switch of alpha subunit carboxyl-terminal domain on an UP-like element.

The interactions between the sigma54-containing RNA polymerase (sigma54-RNAP) and the region of the Pseudomonas putida Pu promoter spanning from the enhancer to the binding site for the integration host factor (IHF) were analyzed both by DNase I and hydroxyl radical footprinting. A short Pu region centered at position -104 was found to be involved in the interaction with sigma54-RNAP, both in the absence and in the presence of IHF protein. Deletion or scrambling of the -104 region strongly reduced promoter affinity in vitro and promoter activity in vivo, respectively. The reduction in promoter affinity coincided with the loss of IHF-mediated recruitment of the sigma54-RNAP in vitro. The experiments with oriented-alpha sigma54-RNAP derivatives containing bound chemical nuclease revealed interchangeable positioning of only one of the two alpha subunit carboxyl-terminal domains (alphaCTDs) both at the -104 region and in the surroundings of position -78. The addition of IHF resulted in perfect position symmetry of the two alphaCTDs. These results indicate that, in the absence of IHF, the sigma54-RNAP asymmetrically uses only one alphaCTD subunit to establish productive contacts with upstream sequences of the Pu promoter. In the presence of IHF-induced curvature, the closer proximity of the upstream DNA to the body of the sigma54-RNAP can allow the other alphaCTD to be engaged in and thus favor closed complex formation.

Base Sequence↗

PubMed2003

Mapping sigma 54-RNA polymerase interactions at the -24 consensus promoter element.

The sigma 54 promoter specificity factor is distinct from sigma 70-type factors. The sigma 54-RNA polymerase binds to promoters with conserved sequence elements at -24 and -12 and utilizes specialized enhancer-binding activators to convert, through an ATP-dependent process, closed promoter complexes to open promoter complexes. The interface between sigma 54-RNA polymerase and promoter DNA is poorly characterized, contrasting with sigma 70. Here, sigma 54 was modified with strategically positioned cleavage reagents to provide physical evidence that the highly conserved RpoN box motif of sigma 54 is close to and may therefore interact with the consensus -24 promoter element. We show that the spatial relationship between the sigma 54-RNA polymerase and the -24 promoter element remains unchanged during closed to open complex conversion and transcription initiation but changes during the early elongation phase. In contrast, the spatial relationship between sigma 54-RNA polymerase and the consensus -12 promoter element changes upon conversion of the closed promoter complex to an open one. We provide evidence that some -12 promoter region-sigma 54 interactions are dependent upon either the core RNA polymerase or a fork junction DNA structure at the -12-position, indicating that DNA fork junctions can substitute for core RNAP. We also show the beta-subunit flap domain contributes to different sets of sigma-promoter DNA interactions at sigma 54- and sigma 70-dependent promoters.

Amino Acid Sequence↗

PubMed2003

Role of a non-natural beta-C-nucleotide unit in DNA as a template for DNA and RNA syntheses and as a substrate for nucleolytic digestion.

A non-natural beta-C-nucleoside bearing a 3,4-dibenzyloxyphenyl group as a nucleobase (X) was synthesized and incorporated into a 34-mer oligomer with the sequence 5'-dTTTTTAAAAAAXATATAGCAGCGACATGTCACCG-3'. This synthetic oligonucleotide was examined for template activity in the enzymatic syntheses of DNA by the Klenow fragments of Escherichia coli DNA polymerase I and the recombinant DNA polymerase I, and in the synthesis of RNA by the E. coli RNA polymerase core enzyme. As a result, the template-directed polymerization of both DNA and RNA was precisely terminated at the position of X. The X-containing oligonucleotide was also tested for digestion by an exonuclease, Exo III nuclease (Exo III), and an endonuclease, Mung Bean nuclease (MB). The results indicate that the artificial nucleobase X acts as a terminator for digestion by Exo III, whereas the site X becomes susceptible to digestion by MB. These findings provide a useful tool for the size control of products in the synthesis and degradation of nucleic acids.

Base Sequence↗

PubMed2003

Two different modes of transcription repression of the Escherichia coli acetate operon by IclR.

IclR is a repressor for the Escherichia coli aceBAK operon, which encodes isocitrate lyase (aceB), malate synthase (aceA) and isocitrate dehydroge-nase kinase/phosphorylase (aceK) in the glyoxylate bypass. IclR also represses the expression of iclR in an autogenous manner. DNase I footprinting and in vitro transcription assays indicated that IclR binds to an IclR box (-21 to +14), which overlaps the iclR promoter and thus competes with the RNA polymerase for DNA binding, leading to transcription repression. In the case of the aceBAK operon, IclR binds to IclR box II between -52 and -19 of the aceB promoter and interferes with binding of the RNA polymerase to this promoter. A secondary IclR binding site (IclR box I) was identified between -125 and -99 of the aceB promoter. IclR binds to this IclR box I even after formation of the aceB promoter open complex and, moreover, induces disassembly of the open complex, leading to repression of aceB transcription. In parallel, the location of the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) on DNA is shifted close to the IclR box I, indicating that direct interaction between the alphaCTD and the IclR box I-associated IclR caused the repression.

Acetates↗

PubMed2003

Identification and molecular characterization of the Mg2+ stimulon of Escherichia coli.

Transcription profile microarray analysis in Escherichia coli was performed to identify the member genes of the Mg(2+) stimulon that respond to the availability of external Mg(2+) in a PhoP/PhoQ two-component system-dependent manner. The mRNA levels of W3110 in the presence of 30 mM MgCl(2), WP3022 (phoP defective), and WQ3007 (phoQ defective) were compared with those of W3110 in the absence of MgCl(2). The expression ratios of a total of 232 genes were <0.75 in all three strains (the supplemental data are shown at http://www.nara.kindai.ac.jp/nogei/seiken/array.html), suggesting that the PhoP/PhoQ system is involved directly or indirectly in the transcription of these genes. Of those, 26 contained the PhoP box-like sequences with the direct repeats of (T/G)GTTTA within 500 bp upstream of the initiation codon. Furthermore, S1 nuclease assays of 26 promoters were performed to verify six new Mg(2+) stimulon genes, hemL, nagA, rstAB, slyB, vboR, and yrbL, in addition to the phoPQ, mgrB, and mgtA genes reported previously. In gel shift and DNase I footprinting assays, all of these genes were found to be regulated directly by PhoP. Thus, we concluded that the phoPQ, mgrB, mgtA, hemL, nagA, rstAB, slyB, vboR, and yrbL genes make up the Mg(2+) stimulon in E. coli.

Bacterial Proteins↗

PubMed2003

Growth phase-coupled alterations in cell structure and function of Escherichia coli.

Escherichia coli cultures can be fractionated into more than 20 cell populations, each having a different bouyant density and apparently representing a specific stage of cell differentiation from exponential growth to stationary phase (H. Makinoshima, A. Nishimura, and A. Ishihama, Mol. Microbiol. 43:269-279, 2002). The density increase was found to be impaired at an early step for a mutant E. coli with the disrupted rpoS gene, which encodes the RNA polymerase RpoS (sigma-S) for stationary-phase gene transcription. This finding suggests that RpoS is need for the entire process of cell density increase. In the absence of RpoF sigma factor, the flagella are not formed as observed by electron microscopy, but the growth phase-coupled density increase takes place as in wild-type E. coli, confirming that the alteration in cell density is not directly correlated with the presence or absence of flagella. In the stationary-phase cells, accumulation of electron-dense areas was observed by electron microscopic observation of bacterial thin sections. By chemical determination, the increase in glycogen (or polysaccharides) was suggested to be one component, which contributes to the increase in weight-to-volume ratio of stationary-phase E. coli cells.

Bacterial Proteins↗

PubMed2003

In vitro properties of RpoS (sigma(S)) mutants of Escherichia coli with postulated N-terminal subregion 1.1 or C-terminal region 4 deleted.

Derivatives of the stationary-phase sigma factor sigma(S) of Escherichia coli lacking either of two conserved domains, the postulated N-terminal subregion 1.1 or the C-terminal region 4, were shown to be competent in vitro for transcription initiation from several sigma(S)-dependent promoters on supercoiled DNA templates. Unlike wild-type sigma(S), however, the deletion derivatives were inactive on relaxed templates. The anomalous slow electrophoretic mobility of sigma(S) on denaturing gels was corrected by deletion of subregion 1.1, suggesting that this domain in sigma(S) may be structurally and functionally analogous to subregion 1.1 of sigma(70), substitutions in which have previously been shown to rectify the anomalous electrophoretic migration of sigma(70) (V. Gopal and D. Chatterji, Eur. J. Biochem. 244:614-618, 1997).

Bacterial Proteins↗

PubMed2003

The fission yeast RPA51 is a functional homolog of the budding yeast A49 subunit of RNA polymerase I and required for maximizing transcription of ribosomal DNA.

Saccharomyces cerevisiae A49 and mouse PAF53 are subunits specific to RNA polymerase I (Pol I) in eukaryotes. It has been known that Pol I without A49 or PAF53 maintains non-specific transcription activities but a molecular role(s) of A49 (and PAF53) remains totally unknown. We studied the fission yeast gene encoding a protein of 415 amino acids exhibiting 30% and 19% identities to A49 and PAF53, respectively. We designate the corresponding protein RPA51 and gene encoding it rpa51+ since the gene encodes a Pol I subunit and an apparent molecular mass of the protein is 51 kDa. rpa51+ is required for cell growth at lower but not at higher temperatures and is able to complement S. cerevisiae rpa49Delta mutation, indicating that RPA51 is a functionally-conserved subunit of Pol I between the budding yeast and the fission yeast. Deletion analysis of rpa51+ shows that only two-thirds of the C-terminal region are required for the function. Transcripts analysis in vivo and in vitro shows that RPA51 plays a general role for maximizing transcription of rDNA whereas it is dispensable for non-specific transcription. We also found that RPA51 associates significantly with Pol I in the stationary phase, suggesting that Pol I inactivation in the stationary phase of yeast does not result from the RPA51 dissociation.

Amino Acid Sequence↗

PubMed2002

Syntheses and structure-activity relationships of nonnatural beta-C-nucleoside 5'-triphosphates bearing an aromatic nucleobase with phenolic hydroxy groups: inhibitory activities against DNA polymerases.

Five nonnatural beta-C-nucleoside 5'-triphosphates bearing a 3,4-dihydroxyphenyl (1TP), a 2-hydroxyphenyl (2TP), a 3-hydroxyphenyl (3TP), a 4-hydroxyphenyl (4TP), or a phenyl (5TP) group were synthesized, and their structure-activity relationships were examined for a series of DNA polymerase reactions in vitro under typical polymerase chain reaction conditions. We found that the 5'-triphosphates (1TP-5TP) are not incorporated into DNA strands but inhibit the DNA polymerase reactions in the presence of natural nucleoside 5'-triphosphates (dNTPs). 1TP having two phenolic hydroxy groups at the nucleobase moiety showed the most potent inhibitory effect against DNA synthesis by Ex Taq polymerase (IC(50) = 30 microM). The competition assay indicated that 1TP and dNTPs are most likely to affect DNA polymerase reactions competitively. This finding may raise the appealing possibility that artificial nucleoside 5'-triphosphates having phenolic hydroxy groups could exhibit potent inhibitory activity against DNA-directed enzymatic reactions.

DNA-Directed DNA Polymerase↗

PubMed2002

Decrease in cell viability in an RMF, sigma(38), and OmpC triple mutant of Escherichia coli.

In a speG-disrupted Escherichia coli mutant, which cannot metabolize spermidine to acetylspermidine, addition of spermidine to the medium caused a decrease in cell viability at the late stationary phase of growth. There were parallel decreases in the levels of ribosome modulation factor (RMF), the sigma(38) subunit of RNA polymerase, and the outer membrane protein C (OmpC). To clarify that these three proteins are strongly involved in cell viability, the rmf, rpoS (encoding sigma(38)), and ompC genes were disrupted. Viability of the triple mutant decreased to less than 1% of normal cells. The triple mutant had a reduced cell viability compared to any combination of double mutants, which also had a reduced cell viability. The single rmf and rpoS, but not ompC, mutant only slightly reduced cell viability. The results indicate that cooperative functions of these three proteins are necessary for cell viability at the late stationary phase. The triple mutant had a reduced level of ribosomes and of intracellular cations.

Bacterial Proteins↗

PubMed2002

Minimum molecular architectures for transcription and replication of the influenza virus.

The RNA-dependent RNA polymerase of influenza virus is composed of three viral P proteins (PB1, PB2, and PA) and involved in both transcription and replication of the RNA genome. The PB1 subunit plays a key role in both the assembly of three P protein subunits and the catalytic function of RNA polymerization. We have established a simultaneous expression system of three P proteins in various combinations using recombinant baculoviruses, and isolated the PA-PB1-PB2 ternary (3P) complex and two kinds of the binary (2P) complex, PA-PB1 and PB1-PB2. The affinity-purified 3P complex showed all of the catalytic properties characteristic of the transcriptase, including capped RNA-binding, capped RNA cleavage, model viral RNA binding, model viral RNA-directed RNA synthesis, and polyadenylation of newly synthesized RNA. The PB1-PB2 binary complex showed essentially the same catalytic properties as does the 3P complex, whereas the PA-PB1 complex catalyzed de novo initiation of RNA synthesis in the absence of primers. Taken together we propose that the catalytic specificity of PB1 subunit is modulated to the transcriptase by binding PB2 or the replicase by interaction with PA.

Base Sequence↗

PubMed2002

Polyamines enhance synthesis of the RNA polymerase sigma 38 subunit by suppression of an amber termination codon in the open reading frame.

The mechanisms by which polyamines stimulate synthesis of the RNA polymerase sigma(38) subunit in Escherichia coli were studied. Polyamine stimulation was observed only in strains in which the 33rd codon of RpoS mRNA is a UAG termination codon instead of a CAG codon for glutamine in wild-type E. coli. Readthrough of the termination codon by Gln-tRNA(supE) was stimulated by polyamines. This stimulation was found to be caused by an increase in both the level of suppressor tRNA(supE) and the binding affinity of Gln-tRNA(supE) for ribosomes. The stimulatory effect was observed with a UAG termination codon but not with UGA and UAA codons. Readthrough of the UAG termination codon at the 270th amino acid position of RpoS mRNA was also stimulated by polyamines, indicating that polyamines stimulate readthrough of a UAG codon regardless of its location within the RpoS mRNA. When cell viability of an E. coli strain having a termination codon in the 33rd position of RpoS mRNA was compared using cells cultured with or without putrescine, it was higher in cells cultured with putrescine than in cells cultured without putrescine. The level of sigma(38) subunit in the cells cultured with putrescine was higher than that in cells cultured without putrescine on days 2, 4, and 8, but the level of sigma(70) subunit was almost the same in cells cultured with or without putrescine. These results confirm that elevated expression of the rpoS gene is important for cell viability at late stationary phase.

Base Sequence↗

PubMed2002

Identification of histone H4-like TAF in Schizosaccharomyces pombe as a protein that interacts with WD repeat-containing TAF.

The general transcription factor TFIID consists of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs). We previously identified two distinct WD repeat-containing TAFs, spTAF72 and spTAF73, in the fission yeast Schizosaccharomyces pombe. Here we report the identification of another S.pombe TAF, spTAF50, which is the S.pombe homolog of histone H4-like TAFs such as human TAF80, Drosophila TAF60 and Saccharomyces cerevisiae TAF60. spTAF50 was identified in a two-hybrid screen as a protein that interacts with the C-terminal WD repeat-containing region of spTAF72. Gene disruption revealed that spTAF50 is essential for cell viability. In vitro, spTAF50 bound to spTAF72 but less efficiently to spTAF73. In S.pombe cells, spTAF50 was detected as a protein with an apparent molecular mass of approximately 50 kDa. Immunoprecipitation experiments demonstrated that spTAF50 is present in both the TFIID and SAGA-like complexes as in the case of spTAF72. These results indicate that the C-terminal region of spTAF72, which largely consists of WD repeats, interacts with spTAF50 in the TFIID and SAGA-like complexes, suggesting a role for the WD repeat domain in the interaction between TAFs.

Amino Acid Sequence↗

PubMed2002

Expression in Escherichia coli and purification of the functional feline granulocyte colony-stimulating factor.

Feline granulocyte colony-stimulating factor (G-CSF) with an N-terminal histidine hexamer tag was expressed as inclusion bodies in E. coli. The G-CSF solubilized in 6 M guanidine solution was absorbed onto a Ni-NTA column and, after washing with decreasing concentrations of guanidine, eluted with imidazole in a soluble and apparently pure form. The activity of the recombinant feline G-CSF was 3 x 10(6)U/mg protein, as assayed by its stimulatory effect on NFS-60 cell proliferation. When a low level of purified feline G-CSF was administered once a day for two successive days to cats, the number of neutrophil increased 4-fold while the levels of other blood cell types remained virtually unchanged. Daily administration of G-CSF for a total of 11 days led to a more than 10-fold increase in neutrophils, an 8-fold increase in the number of monocytes and 2-fold increase in lymphocytes. No severe side effects or antibody production was observed in cats after administration of G-CSF.

Animals↗

PubMed2002

The interaction between sigmaS, the stationary phase sigma factor, and the core enzyme of Escherichia coli RNA polymerase.

BACKGROUND: The RNA polymerase holoenzyme of Escherichia coli is composed of a core enzyme (subunit structure alpha2betabeta') associated with one of the sigma subunits, required for promoter recognition. Different sigma factors compete for core binding. Among the seven sigma factors present in E. coli, sigma70 controls gene transcription during the exponential phase, whereas sigmaS regulates the transcription of genes in the stationary phase or in response to different stresses. Using labelled sigmaS and sigma70, we compared the affinities of both sigma factors for core binding and investigated the structural changes in the different subunits involved in the formation of the holoenzymes. RESULTS: Using native polyacrylamide gel electrophoresis, we demonstrate that sigmaS binds to the core enzyme with fivefold reduced affinity compared to sigma70. Using iron chelate protein footprinting, we show that the core enzyme significantly reduces polypeptide backbone solvent accessibility in regions 1.1, 2.5, 3.1 and 3.2 of sigmaS, while increasing the accessibility in region 4.1 of sigmaS. We have also analysed the positioning of sigmaS on the holoenzyme by the proximity-dependent protein cleavage method using sigmaS derivatives in which FeBABE was tethered to single cysteine residues at nine different positions. Protein cutting patterns are observed on the beta and beta' subunits, but not alpha. Regions 2.5, 3.1 and 3.2 of sigmaS are close to both beta and beta' subunits, in agreement with iron chelate protein footprinting data. CONCLUSIONS: A comparison between these results using sigmaS and previous data from sigma70 indicates similar contact patterns on the core subunits and similar characteristic changes associated with holoenzyme formation, despite striking differences in the accessibility of regions 4.1 and 4.2.

Bacterial Proteins↗

PubMed2002

Level of the RNA polymerase II in the fission yeast stays constant but phosphorylation of its carboxyl terminal domain varies depending on the phase and rate of cell growth.

BACKGROUND: The RNA polymerase II of the fission yeast Schizosaccharomyces pombe consists of 12 Rpb subunits, of which four (Rpb1, Rpb2, Rpb3 and Rpb11) form the assembly and catalytic core and five (Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12) are shared among RNA polymerases I, II and III. The intracellular levels of three RNA polymerase forms should be interrelated, but the control of RNA polymerase formation remains mostly unknown. RESULTS: To reveal the physiological role and the synthesis control of each Rpb subunit, the intracellular levels of the Rpb proteins were examined in S. pombe growing at various phases under various conditions. Results indicate that the intracellular concentrations of the Rpb proteins stay constant at levels characteristic of the rate and phase of cell growth, and the relative level between the 12 subunits also remains constant, together implying that the intracellular concentration of RNA polymerase II stays constant, as in the case of prokaryotes. As an attempt to gain insights into the activity control of RNA polymerase II, we also analysed the phosphorylation level of the carboxyl-terminal domain (CTD) of the largest subunit Rpb1. Phosphorylated forms of Tyr1 and Thr4 within 29 repeats of the YSPTSPS heptapeptide were detected in both slow-migrating IIo and fast-migrating IIa forms of Rpb1 on SDS-PAGE (polyacrylamide gel electrophoresis). However, phosphorylated Ser2 and Ser5 were identified only in the IIo form, indicating that Ser phosphorylation contributes to the conformational change in CTD. The phosphorylation levels of Ser, Thr and Tyr all vary depending on the cell culture conditions. CONCLUSION: The intracellular level of RNA polymerase II stays constant, but the amount engaged in transcription cycle varies depending on the culture conditions, as estimated from the sites and levels of phosphorylation of Rpb1 CTD.

Phosphorylation↗

PubMed2002

Fractionation of Escherichia coli cell populations at different stages during growth transition to stationary phase.

Cultures of Escherichia coli could be separated into more than 15 cell populations, each forming a discrete band after Percoll gradient centrifugation. The cell separation was found to result from the difference in buoyant density but not the size difference. The cell density increases upon transition from exponential growth to stationary phase. Exponential phase cultures formed at least five discrete bands with lower densities, whereas stationary phase cultures formed more than 10 bands with higher densities. Two molecular markers characterizing each cell population were identified: the functioning promoter species, as identified by measuring the expression of green fluorescent protein under the control of test promoters; and the expressed protein species, as monitored by quantitative immunoblotting. These findings together suggest that the growth phase-coupled transition of E. coli phenotype is discontinuous.

Bacterial Proteins↗