N Fujita

National Institute of Genetics, Мисима, Shizuoka, Japan

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Publications (117)482.05 Total impact

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    ABSTRACT: The expression pattern of the Escherichia coli genome is controlled in part by regulating the utilization of a limited number of RNA polymerase among a total of its approximately 4,600 genes. The distribution pattern of RNA polymerase changes from modulation of two types of protein-protein interactions: the interaction of core RNA polymerase with seven species of the sigma subunit for differential promoter recognition; and the interaction of RNA polymerase holoenzyme with about 300 different species of transcription factors (TFs) with regulatory functions. We have been involved in the systematic search for the target promoters recognized by each sigma factor and each TF using the newly developed Genomic SELEX system. In parallel, we developed the promoter-specific (PS)-TF screening system for identification of the whole set of TFs involved in regulation of each promoter. Understanding the regulation of genome transcription also requires knowing the intracellular concentrations of the sigma subunits and TFs under various growth conditions. This report describes the intracellular levels of 65 species of TF with known function in E. coli K-12 W3110 at various phases of cell growth and at various temperatures. The list of intracellular concentrations of the sigma factors and TFs provides a community resource for understanding the transcription regulation of E. coli under various stressful conditions in nature.
    Journal of bacteriology. 05/2014;
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    ABSTRACT: CRP (cAMP receptor protein), the global regulator of genes for carbon source utilization in the absence of glucose, is the best-studied prokaryotic transcription factor. A total of 195 target promoters on the Escherichia coli genome have been proposed to be under the control of cAMP-bound CRP. Using the newly developed Genomic SELEX screening system of transcription factor-binding sequences, however, we have identified a total of at least 254 CRP-binding sites. Based on their location on the E. coli genome, we predict a total of at least 183 novel regulation target operons, altogether with the 195 hitherto known targets, reaching to the minimum of 378 promoters as the regulation targets of cAMP-CRP. All the promoters selected from the newly identified targets and examined by using the lacZ reporter assay were found to be under the control of CRP, indicating that the Genomic SELEX screening allowed to identify the CRP targets with high accuracy. Based on the functions of novel target genes, we conclude that CRP plays a key regulatory role in the whole processes from the selective transport of carbon sources, the glycolysis-gluconeogenesis switching to the metabolisms downstream of glycolysis, including tricarboxylic acid (TCA) cycle, pyruvate dehydrogenase (PDH) pathway and aerobic respiration. One unique regulation mode is that a single and the same CRP molecule bound within intergenic regions often regulates both of divergently transcribed operons.
    PLoS ONE 01/2011; 6(6):e20081. · 3.53 Impact Factor
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    ABSTRACT: In Escherichia coli, CitA is a membrane-associated sensor histidine kinase that phosphorylates CitB, the response regulator. It is predicated to play a key role in anaerobic citrate catabolism. The citrate-binding site in CitA is located within its periplasmic domain, while the cytoplasmic domain (CitA-C) is involved in autophosphorylation. We found that autophosphorylation in vitro of CitA-C was induced by DTT. Using the whole set of CitA-C derivatives containing Cys-Ala substitution(s), Cys at 529 was found to be essential to the redox-sensing of autophosphorylation. The phosphorylated CitA-C transferred a phosphate to CitB. DNase-I footprinting assay indicated that CitB specifically bound on the intergenic region between the citA and citC genes. These results characterize the molecular mechanism of the CitA-CitB signal transduction system in E. coli.
    Bioscience Biotechnology and Biochemistry 03/2009; 73(2):346-50. · 1.27 Impact Factor
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    ABSTRACT: The pattern of genome transcription in prokaryotes is determined by selective distribution of RNA polymerase within the genome. The gene selectivity of RNA polymerase is modulated after interaction with DNA-binding transcription factors (TFs). Escherichia coli contains a total of about 300 TFs, but the regulatory function has not yet been identified for about one third. For quick and systematic search of the regulation target genes by TFs, we have developed a novel screening technology ¿Genomic SELEX¿, in which the recognition DNA sequences by the test TF are isolated from a mixture of genome DNA fragments. The sequences were determined by either cloning-sequencing of SELX fragments (SELEX-clos) or high-density microarray (tilling array) analysis (SELEX-chip). Here we describe the application of these novel technologies for search of the whole set of regulation target genes on the E. coli genome by CRP (cAMP-binding protein).
    01/2009;
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    ABSTRACT: In Escherichia coli, CitB, a cognate response regulator of CitA, specifically bound to the promoter regions for mdh, citA, citC, and exuT. Transcription of these genes was induced by citrate under anaerobic conditions in a CitAB-dependent manner. Taking this together, we conclude that CitAB is the master regulatory system that activates the set of genes involved in citrate fermentation in E. coli.
    Bioscience Biotechnology and Biochemistry 12/2008; 72(11):3011-4. · 1.27 Impact Factor
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    ABSTRACT: The sigma 28 kDa (sigma28) factor is a transcription factor specific for the expression of bacterial flagellar and chemotaxis genes. Its antisigma factor, FlgM, binds sigma28 factor and inhibits its activity as a transcription factor. In this study, crystals of the complex between Escherichia coli sigma28 and the C-terminal sigma28-binding region of FlgM were obtained. The crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 106.7 (2), c = 51.74 (3) A, containing one complex in the crystallographic asymmetric unit. An X-ray intensity data set was collected to a resolution of 2.7 A.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 04/2007; 63(Pt 3):196-9. · 0.55 Impact Factor
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    ABSTRACT: The effects of a number of mutations in crp have been measured at different cyclic AMP receptor protein (CRP)-dependent Class II promoters, where the CRP-binding site is centred around 411/2 base pairs upstream from the transcription start point. The amino acid substitutions HL159 and TA158 result in reduced CRP-dependent activation, but the reduction varies from one Class II promoter to another. Deletions in the C-terminus of the RNA polymerase alpha subunit suppress the effects of HL159 and TA158. The role of the C-terminus of alpha at these promoters is assessed. Other changes at E58, K52 and E96 affect CRP activity specifically at Class II promoters and their role is discussed.
    Molecular Microbiology 10/2006; 10(4):789 - 797. · 5.03 Impact Factor
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    01/2006;
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    ABSTRACT: Cra (or FruR), a global transcription factor with both repression and activation activities, controls a large number of the genes for glycolysis and gluconeogenesis. To get insights into the entire network of transcription regulation of the E. coli genome by Cra, we isolated a set of Cra-binding sequences using an improved method of genomic SELEX. From the DNA sequences of 97 independently isolated DNA fragments by SELEX, the Cra-binding sequences were identified in a total of ten regions on the E. coli genome, including promoters of six known genes and four hitherto-unidentified genes. All six known promoters are repressed by Cra, but none of the activation-type promoters were cloned after two cyles of SELEX, because the Cra-binding affinity to the repression-type promoters is higher than the activation-type promoters, as determined by the quantitative gel shift assay. Of a total of four newly identified Cra-binding sequences, two are associated with promoter regions of the gapA (glyceraldehyde 3-phosphate dehydrogenase) and eno (enolase) genes, both involved in sugar metabolism. The regulation of newly identified genes by Cra was confirmed by the in vivo promoter strength assay using a newly developed TFP (two-fluorescent protein) vector for promoter assay or by in vitro transcription assay in the presence of Cra protein.
    Genes to Cells 10/2005; 10(9):907-18. · 2.73 Impact Factor
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    ABSTRACT: Immunoblotiing of size- seperated whole cell protein permitted the study of protein-protein interaction. Briefly, protein obtained from cleared cell lysates of Escherichia coli were seperated by glycerol gradient centrifugation and analysed by blooting against a set of specific antibodies. We have applied this procedure to the assembly of 11 N-terminla amber fragment of the β subunit of E. coli RNA polymerase ranging in size between 97% and 23% the lenght of the intact β polypeptide (1342 amino acids). In this way, we have been able to define regions on the β polypeptide involved in the assembly of RNA polymerase.
    European Journal of Biochemistry. 03/2005; 176(2):403 - 407.
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    ABSTRACT: PhoP is a response regulator of the PhoQ-PhoP two-component system controlling a set of the Mg(II)-response genes in Escherichia coli. Here we demonstrate the mode of transcription regulation by phosphorylated PhoP of divergently transcribed mgtA and treR genes, each encoding a putative Mg(II) transporter and a repressor for the trehalose utilization operon respectively. Under Mg(II)-limiting conditions in vivo, two promoters, the upstream constitutive P2 and the downstream inducible P1, were detected for the mgtA gene. Gel-shift analysis in vitro using purified PhoP indicates its binding to a single DNA target, centred between -43 and -24 of the mgtAP1 promoter. This region includes the PhoP box, which consists of a direct repeat of the heptanucleotide sequence (T)G(T)TT(AA). Site-directed mutagenesis studies indicate the critical roles for T (position 3), T (position 4) and A (position 6) for PhoP-dependent transcription from mgtAP1. DNase I footprinting assays reveal weak binding of PhoP to this PhoP box, but the binding becomes stronger in the simultaneous presence of RNA polymerase. Likewise the RNA polymerase binding to the P1 promoter becomes stronger in the presence of PhoP. For the PhoP-assisted formation of open complex at the mgtAP1 promoter, however, the carboxy-terminal domain of alpha subunit (alpha CTD) is not needed. For transcription in vivo of the treR gene, four promoters were identified. The most upstream promoter treRP4 divergently overlaps with the mgtAP1 promoter, sharing the same sequence as the respective -10 signal in the opposite direction. In vitro transcription using mutant promoters support this prediction. In the presence of PhoP, transcription from the promoter treRP3 was repressed with concomitant activation of mgtAP1 transcription. The PhoP box is located between -46 and -30 with respect to treRP3, and the alpha CTD is needed for this repression.
    Molecular Microbiology 08/2002; 45(2):423-38. · 5.03 Impact Factor
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    ABSTRACT: 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. 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. 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.
    Genes to Cells 04/2002; 7(3):233-47. · 2.73 Impact Factor
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    O N Ozoline, N Fujita, A Ishihama
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    ABSTRACT: The C-terminal domain (CTD) downstream from residue 235 of Escherichia coli RNA polymerase alpha subunit is involved in recognition of the promoter UP element. Here we have demonstrated, by DNase I and hydroxyl radical mapping, the presence of two UP element subsites on the promoter D of phage T7, each located half and one-and-a-half helix turns, respectively, upstream from the promoter -35 element. This non-typical UP element retained its alphaCTD-binding capability when transferred into the genetic environment of the rrnBP1 basic promoter, leading to transcription stimulation as high as the typical rrnBP1 UP element. Chemical protease FeBABE conjugated to alphaCTD S309C efficiently attacked the T7D UP element but not the rrnBP1 UP element. After alanine scanning, most of the amino acid residues that were involved in rrnBP1 interaction were also found to be involved in T7D UP element recognition, but alanine substitution at three residues had the opposite effect on the transcription activation between rrnBP1 and T7D promoters. Mutation E286A stimulated T7D transcription but inhibited rrnBP1 RNA synthesis, while L290A and K304A stimulated transcription from rrnBP1 but not the T7D promoter. Taken together, we conclude that although the overall sets of amino acid residues responsible for interaction with the two UP elements overlap, the mode of alphaCTD interaction with T7D UP element is different from that with rrnBP1 UP element, involving different residues on helices III and IV.
    Nucleic Acids Research 01/2002; 29(24):4909-19. · 8.81 Impact Factor
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    ABSTRACT: In the deoP2 promoter of Escherichia coli, a transcription activator, cAMP-CRP, binds at two sites, centered at -41.5 and -93.5 from the start site of transcription, while a repressor, CytR, binds to a space between the two cAMP-CRP complexes. The mechanisms for the cAMP-CRP-mediated transcription activation and CytR-mediated transcription repression were investigated in vitro using purified components. We classified the deoP2 promoter as a class II cAMP-CRP-dependent promoter, primarily by the action of cAMP-CRP at the downstream site. Interestingly, we also found that deoP2 carries an "UP-element" immediately upstream of the downstream cAMP-CRP site. The UP-element overlaps with the DNA site for CytR. However, it was observed that CytR functions with the RNA polymerase devoid of the C-terminal domain of the alpha-subunit as well as with intact RNA polymerase. The mechanism of repression by CytR proposed in this study is that the cAMP-CRP bound at -41.5 undergoes an allosteric change upon direct interaction with CytR such that it no longer maintains a productive interaction with the N-terminal domain of alpha, but instead acts as a repressor to interfere with RNA polymerase acting on deoP2.
    The EMBO Journal 11/2001; 20(19):5392-9. · 9.82 Impact Factor
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    ABSTRACT: SdiA, an Escherichia coli homologue of the quorum-sensing regulator, controls the expression of the ftsQAZ operon for cell division. Transcription of ftsQ is under the control of two promoters, upstream ftsQP2 and downstream ftsQP1, which are separated by 125 bp. SdiA activates transcription from ftsQP2 in vivo. Here, we demonstrate that SdiA facilitates the RNA polymerase binding to ftsQP2 and thereby stimulates transcription from P2. Gel shift and DNase I footprinting assays indicated that SdiA binds to the ftsQP2 promoter region between -51 and -25 with respect to the P2 promoter. Activation of ftsQP2 transcription by SdiA was observed with a mutant RNA polymerase containing a C-terminal domain (CTD)-deleted alpha-subunit (alpha 235) but not with RNA polymerase containing sigma(S) or a CTD-deleted sigma(D) (sigma(D)529). In good agreement with the transcription assay, no protection of P2 was observed with the RNA polymerase holoenzymes, E sigma(S) and E sigma(D)529. These observations together indicate that: (i) SdiA supports the RNA polymerase binding to ftsQP2; and (ii) this recruitment of RNA polymerase by SdiA depends on the presence of intact sigmaCTD. This is in contrast to the well-known mechanism of RNA polymerase recruitment by protein-protein contact between class I factors and alpha CTD. In addition to the P2 activation, SdiA inhibited RNA polymerase binding to the ftsQP1 promoter and thereby repressed transcription from P1. Gel shift assays indicate weak binding of SdiA to the P1 promoter region downstream from -13 (or +112 with respect to P2). Neither alpha CTD nor sigma CTD are required for this inhibition. Thus, the transcription repression of P1 by SdiA may result from its competition with the RNA polymerase in binding to this promoter.
    Molecular Microbiology 10/2001; 41(5):1187-98. · 5.03 Impact Factor
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    H Maeda, N Fujita, A Ishihama
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    ABSTRACT: Seven different species of the RNA polymerase sigma subunit exist in Escherichia coli, each binding to a single species of the core enzyme and thereby directing transcription of a specific set of genes. To test the sigma competition model in the global regulation of gene transcription, all seven E.coli sigma subunits have been purified and compared for their binding affinities to the same core RNA polymerase (E). In the presence of a fixed amount of sigma(70), the principal sigma for growth-related genes, the level of Esigma(70) holoenzyme formation increased linearly with the increase in core enzyme level, giving an apparent K:(d) for the core enzyme of 0.26 nM. Mixed reconstitution experiments in the presence of a fixed amount of core enzyme and increasing amounts of an equimolar mixture of all seven sigma subunits indicated that sigma(70) is strongest in terms of core enzyme binding, followed by sigma(N), sigma(F), sigma(E)/sigma(FecI), sigma(H) and sigma(S) in decreasing order. The orders of core binding affinity between sigma(70) and sigma(N) and between sigma(70) and sigma(H) were confirmed by measuring the replacement of one core-associated sigma by another sigma subunit. Taken together with the intracellular sigma levels, we tried to estimate the number of each holoenzyme form in growing E. coli cells.
    Nucleic Acids Research 10/2000; 28(18):3497-503. · 8.81 Impact Factor
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    ABSTRACT: sigma(38) (or sigma(S), the rpoS gene product) is a sigma subunit of RNA polymerase in Escherichia coli and directs transcription from a number of stationary-phase promoters as well as osmotically inducible promoters. In this study, we analyzed the function of the carboxy-terminal 16-amino-acid region of sigma(38) (residues 315 to 330), which is well conserved among the rpoS gene products of enteric bacterial species. Truncation of this region was shown to result in the loss of sigma activity in vivo using promoter-lacZ fusion constructs, but the mutant sigma(38) retained the binding activity in vivo to the core enzyme. The in vitro transcription analysis revealed that the transcription activity of sigma(38) holoenzyme under high potassium glutamate concentrations was significantly decreased by the truncation of the carboxy-terminal tail element.
    Journal of Bacteriology 09/2000; 182(16):4628-31. · 3.19 Impact Factor
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    ABSTRACT: Two distinct classes of RNA polymerase sigma factors (sigma) exist in bacteria and are largely unrelated in primary amino acid sequence and their modes of transcription activation. Using tethered iron chelate (Fe-BABE) derivatives of the enhancer-dependent sigma(54), we mapped several sites of proximity to the beta and beta' subunits of the core RNA polymerase. Remarkably, most sites localized to those previously identified as close to the enhancer-independent sigma(70) and sigma(38). This indicates a common use of sets of sequences in core for interacting with the two sigma classes. Some sites chosen in sigma(54) for modification with Fe-BABE were positions, which when mutated, deregulate the sigma(54)-holoenzyme and allow activator-independent initiation and holoenzyme isomerization. We infer that these sites in sigma(54) may be involved in interactions with the core that contribute to maintenance of alternative states of the holoenzyme needed for either the stable closed promoter complex conformation or the isomerized holoenzyme conformation associated with the open promoter complex. One site of sigma(54) proximity to the core is apparently not evident with sigma(70), and may represent a specialized interaction.
    The EMBO Journal 07/2000; 19(12):3038-48. · 9.82 Impact Factor
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    ABSTRACT: To test whether OmpR is involved in regulation of the bolA1p, we investigated possible effects of ompR mutation on transcription from bolA1p. In vivo, bolA1p was found to be repressed by OmpR. Furthermore in vitro, the phospho-OmpR was found to bind to the OmpR binding region of bolA1p and repress the transcription by Esigma(S) or Esigma(D). These results suggest that the phosphorylated form of OmpR is a negative regulator for the transcription of the bolA1p promoter.
    FEMS Microbiology Letters 06/2000; 186(2):257-62. · 2.05 Impact Factor
  • N Fujita, S Endo, A Ishihama
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    ABSTRACT: The carboxy-terminal domain of the alpha subunit of Escherichia coli RNA polymerase, which is connected with the core part of RNA polymerase through a long flexible linker, plays decisive roles in transcription activation by directly interacting with a large number of transcription factors and upstream (UP) element DNA. Here we constructed a set of mutant RNA polymerases, each containing a mutant alpha subunit with an altered interdomain linker. Deletion of three amino acids from the linker exhibited 50% inhibition of cAMP receptor protein- (CRP-) dependent lac P1 transcription. Deletion of six amino acids completely knocked out the activity. Insertion of three amino acids did not affect the activity, whereas 40-60% inhibition was observed after insertion of one, two, or four amino acids. Substitution of 10 consecutive glycine residues resulted in nearly 90% reduction of the CRP-dependent activity, whereas 50% activity was retained after substitution of 10 proline residues or a sequence expected to form a strong alpha-helix. Essentially the same results were obtained with UP element-dependent rrnB P1 transcription. These observations altogether suggest that (i) sufficient length of the interdomain linker is required for transcription activation mediated by the alpha carboxy-terminal domain, (ii) the linker is not totally unstructured but has structural and torsional preferences to facilitate positioning of the carboxy-terminal domain to a proper location for the interaction with CRP and UP element, and (iii) CRP-dependent activation and UP element-dependent activation share a common intermediary state in which the positioning of the alpha carboxy-terminal domain is of primary importance.
    Biochemistry 06/2000; 39(20):6243-9. · 3.38 Impact Factor

Publication Stats

3k Citations
482.05 Total Impact Points

Institutions

  • 1986–2014
    • National Institute of Genetics
      • Division of Molecular Genetics
      Мисима, Shizuoka, Japan
    • University of Cambridge
      • Department of Pathology
      Cambridge, ENG, United Kingdom
  • 2009–2011
    • Hosei University
      • Department of Frontier Bioscience
      Tokyo, Tokyo-to, Japan
  • 2005–2011
    • National Institute of Technology and Evaluation
      Edo, Tōkyō, Japan
  • 2008
    • Kinki University
      Ōsaka, Ōsaka, Japan
  • 1993–2000
    • The University of Tokyo
      • • Institute of Molecular and Cellular Biosciences
      • • Radioisotope Center
      Tokyo, Tokyo-to, Japan
  • 1998
    • University of Rochester
      • Department of Biology
      Rochester, NY, United States
  • 1996
    • University of Maryland, Baltimore County
      • Department of Biological Sciences
      Baltimore, MD, United States
  • 1992–1994
    • The University of Edinburgh
      • Institute of Cell Biology
      Edinburgh, SCT, United Kingdom
  • 1990–1994
    • Kyoto University
      • • Institute for Virus Research
      • • Department of Physics II
      Kioto, Kyōto, Japan
  • 1988
    • Nagoya University
      Nagoya, Aichi, Japan