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1 Nippon Institute for Biological Science, Division of Molecular Biology, Ome, Tokyo 198-0024, Japan
2 Meiji University, Faculty of Agriculture, Kawasaki, Kanagawa 214-8571, Japan
3 National Institute of Technology and Evaluation, Genome Analysis Center, Nishihara, Shibuya, Tokyo 151-0066, Japan
4 Hosei University, Faculty of Engineering and Research Center for Micro-Nano Technology, Koganei 184-8584, Tokyo 184-8584, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Cra was initially characterized as the fructose repressor (FruR), but was later recognized as a pleiotropic regulator that controls transcription of the genes in major pathways for carbon and energy metabolism (Ramseier et al. 1993, 1995; Ramseier 1996; Saier 1996; Saier & Ramseier 1996; Saier et al. 1996). For instance, the genes for sugar catabolism such as fruB (fructose-specific PTS), pfkA (6-phosphofructokinase I) and pykF (pyruvate kinase I) are repressed by Cra while the genes for glyconeogenesis such as ppsA (phosphoenolpyruvate synthase) and fbp (fructose-1,6-bisphosphatase) are activated by Cra (Chin et al. 1989; Ramseier 1996). Derepression of the genes for glycolysis takes place when Cra is inactivated after interaction with inducers (or effectors) such as D-fructose-1-phosphate and D-fructose-1,6-bisphosphate. On the other hand, activation of the genes for gluconeogenesis is associated with an increase in cellular Cra levels (Ramseier et al. 1993). Cra also regulates the equilibrium reaction of glycolytic pathway by repressing the dicistronic gapB(epd)-pgk operon (Charpentier et al. 1998) that encode D-erythrose 4-phosphate dehydrogenase and phosphoglycerate kinase.
Previously, Negre et al. (1996) performed a systematic search for the Cra-binding sequences from a pool of synthetic oligonucleotides with all possible sequences using the CAST (cyclic amplification and selection of targets) procedure, which was developed by Norby et al. (1992) and Wright & Funk (1993) [note that CAST is essentially the same approach as SELEX (systematic evolution of ligands by exponential enrichment) (Tuerk & Gold 1990)], and identified 20 imperfect palindromic DNA sequences, indicating that the Cra tetramer interacts asymmetrically with two half-sites. After a computer-based homology search for the consensus sequence, the pfkA gene, coding for phosphofructokinase-1, was added as a target of Cra. The CAST approaches have proved to be useful in searching for the recognition sequences of DNA-binding proteins from pools of random sequences. In the case of DNA-binding transcription factors, however, it is sometimes difficult to identify the whole set of target genes from the entire genome, because of the variation among the sequences recognized by a single factor, with different affinities to the test factor. Bacteria use this sequence variation (and thus the affinity difference) within sets of regulated genes to establish different levels of expression between genes in the same regulon.
In order to establish an experimental system to reveal the entire regulatory network on the E. coli genome by any test transcription factor, we employed genomic SELEX (Singer et al. 1997), which uses a complete library of genome DNA fragments, instead of synthetic DNA with all possible sequences. Here we applied this improved genomic SELEX for searching the whole set of natural Cra-binding DNA sequences on the E. coli genome. So far, we have identified four new Cra target sequences, including two genes (eno and gapA) with known functions, and two unidentified genes (hypF and yahA). Regulation of the eno and gapA genes by Cra was confirmed in both in vivo and in vitro transcription assays. After measurement of the binding affinity of Cra to the target DNA sequences, we concluded that the Cra-binding affinity was higher for the repressing targets than for activating targets.
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Results |
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In the original SELEX procedure, target DNA sequences for a test DNA-binding protein are isolated from mixtures of synthetic oligonucleotides with all possible sequences (Tuerk & Gold 1990). The screening is based on binding a test protein and partitions high-affinity sequences from low-affinity sequences. Using this procedure, however, it is difficult to isolate target DNA sequences if the test protein recognizes a signal consisting of separated DNA sequences. Even if the test protein recognizes a continuous sequence, it is also difficult to prepare high-concentration solutions of mixed DNA of all possible sequences for detection of complexes with proteins of low DNA-binding affinity. To overcome this difficulty, we have developed an improved genomic SELEX system by using a mixture of genome DNA fragments. We first prepared a plasmid library of size-fractionated DNA fragments of the E. coli genome. The library herein used contained about 105 independent plasmid clones, each carrying a specific DNA fragment of 100–300 bp in length, that was generated by sonication of the E. coli genome (N. Fujita and S. Endo, unpublished observation). The combination of all E. coli DNA fragments in this library corresponds to a total DNA sequence of about 5.43 times the total E. coli genome. This library was used to search for DNA sequences recognized by Cra, a global regulator of genes involved in carbon metabolism.
For isolation of DNA sequences that are recognized by E. coli Cra protein, genomic DNA fragments were re-generated by PCR using a pair of primers, which bind to the vector sequences at the 5' and 3' vector-insert junctions. Purified His-tagged Cra protein was mixed with two-fold molar excess of the DNA fragment mixture, and Cra-DNA complexes were isolated by Ni-NTA column chromatography. The DNA fragments thus isolated, hereafter referred to as ‘SELEX fragments’, were recovered from Cra-complexes and directly sequenced using the same set of primers, which were used for the generation of SELEX fragments. After one cycle of this SELEX, the recovered DNA fragments formed smear on PAGE. DNA samples recovered from the smear included various sequence with or without Cra box. The SELEX cycle was then repeated 4–5 times, but the number of different Cra-binding DNA sequences decreased after the third cycle (data not shown). Thus, for random screening of as many Cra-binding sequences as possible, and minimizing the background noise, we isolated a total 100 independent DNA fragments after two cycles of the genomic SELEX. After DNA sequencing of these SELEX fragments, a total of 97 sequences, that located at 11 regions of the E. coli genome, was found. One SELEX fragment, which included a part of the mppA-coding sequence, formed a complex with Cra but a Cra-binding sequence could not be identified in this fragment. Apart from this mppA fragment, detailed analysis was then performed for ten different SELEX fragments. Table 1 shows the genes adjacent to each SELEX fragment and the direction of transcription of the neighboring genes. On the basis of the gene orientation, all 10 SELEX fragments were predicted to include the signal sequences to control expression of the respective downstream genes (Fig. 1).
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Identification of Cra-binding activity for DNA sequences isolated by SELEX
To identify the binding activity of Cra for all 11 SELEX fragments (6 known and 5 previously unidentified, including mppA), we carried out DNA mobility shift assays. Based on the sequences of the SELEX fragments, we prepared DNA fragments, containing the promoter region starting from the respective initiation codon and including the respective SELEX fragment sequence (see Fig. 1). In the case of mppA, the probe was designed so as to include a part of the protein-coding sequence. The gel shift assay was performed using increasing concentrations of the purified Cra protein. The amounts of unbound free and protein-bound DNA probes were determined by measuring the intensity of autoradiograms. Results, summarized in Fig. 2A, indicated: (1) ten fragments including all six known Cra-regulated promoters formed Cra complexes; (2) four newly identified SELEX fragments (gapA, eno, yahA and hypF) indeed formed Cra complexes; (3) two promoter fragments, fruB and gapA, gave two shift bands, implying the binding of two Cra tetramers on these fragments; and (4) the mppA coding sequence did not form Cra complex. The formation of two shift bands by fruB is in good agreement of the presence of two Cra-binding sequences on the fruB promoter (Ramseier et al. 1993). Two separate Cra-binding sites were also detected for the gapA promoter (see below).
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Consensus sequence for Cra binding
From the ten Cra-binding sequences (six newly identified in this study and seven known Cra-box sequences), we can propose a complete palindromic sequence, TGAAAC-GTTTCA, with an extended GC at the 5' end, for the typical Cra box (Fig. 3). This is consistent with the proposed Cra-binding seqeuence RSTGAAWCSNTHHW (Ramseier et al. 1993, 1995; Negre et al. 1996). Using this consensus sequence, we searched for the Cra-binding site on the entire E. coli genome. Most of the genes carrying the Cra-box sequence at their promoter regions (upstream from the coding sequences) are included in the collection herein identified. Although Cra box-like sequences exist upstream from promoters of the adk, aldH, degP, infC, pasB, rseB, sdaB, and sodC genes (data not shown), it is unlikely that the Cra box-like sequences play regulatory roles in transcription of these genes because these sequences are located far from the respective initiation codons. Cra box-like sequences also exist within the coding regions of some other genes, besides the mppA gene, including the the alsB, garD, hchA, mesJ, pepD, ppx, ptsA, rspB, sun, and rig genes. Possible roles of these Cra box-like sequences within the protein-coding sequences remain a mystery.
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Using the quantitative gel shift assay (Talukder & Ishihama 1999), we next measured the dissociation constant (Kd) for all the test DNA fragments. The apparent Kd was calculated as the protein concentration, which gave to shift 50% of the input DNA into Cra complexes (Fig. 2C). The affinity to Cra is generally higher for the promoters repressed by Cra, while in the absence of inducers (or effectors), the promoters from the genes activated by Cra showed low affinity to Cra protein. Repression of transcription in vitro of the repressor-type promoters was also observed at lower concentrations of Cra than that used for transcription activation of the activation-type promoters (Prost et al. 1999; T. Shimada, unpublished observation).
The Cra-binding affinity is the highest (apparent Kd = 1.9 x 10–6 m) for the fruB promoter, which carries two Cra-box sequences, in agreement with the finding that the fruB promoter is the most abundant SELEX fragment, i.e. 43 copies (44.3%) among a total of 97 sequenced fragments. In contrast, the Cra-binding affinity was among the weakest group for two repression-type promoters, edd (Kd = 12.4 x 10–6 M) and pfkA (Kd = 12.8 x 10–6 M), which were not isolated in our SELEX screening. The three newly identified promoters, yahA, gapA and eno, with Cra-binding site showed high affinity to Cra (Kd for yahA = 2.4 x 10–6 M; Kd for gapA = 3.0 x 10–6 M; and Kd for eno = 6.2 x 10–6 M), suggesting that these three promoters belong to the repression-type group. One newly identified promoter, hypF, could be classified at the border between the repression- and activation-type promoters with respect to the apparent Kd value.
Under the genomic SELEX conditions employed (protein/DNA input molar ratio of 2, two cycles of SELEX and in the absence of inducers), we failed to detect the promoters (icdA, aceB, ppsA, acnA and pckA), which are known to be activated by Cra. We then prepared the Cra box-containing DNA fragments from these activation-type promoters, and measured the Cra-binding affinity in the absence of inducers. The apparent Kd value for these promoters were found to be above 17.4 x 10–6M (Fig. 2C). We then concluded that DNA fragments with the Kd value for Cra-binding below 10–5M could be isolated under the SELEX conditions employed.
Transcription regulation of the gapA gene by Cra
After the genomic SELEX screening, we identified four new sequences with Cra-binding activity, two genes (gapA and eno) with known functions and two unidentified genes (yahA and hypF). The Cra-binding activity was detected for all these fragments by the gel shift assay (Figs 2 and 3). First, detailed analysis of transcription regulation was then carried out for two known genes, gapA (glyceraldehyde 3-phosphate dehydrogenase) and eno (enolase), with known functions. To identify the Cra-binding sites on the gapA promoter region, we carried out the gel shift assay using one full-length F12 DNA probe (968 bp from –1 to –968 bp from the initiation codon) and two separated DNA fragments (500 bp-long F1 from –1 to –500 bp, and 506 bp-long F2 from –463 to –968 bp) (Fig. 4A). The full-length probe gave two gel shifted bands (Fig. 4B; also see Fig. 2A), while both F1 and F2 fragments formed one shift band (Fig. 4B). Results indicate the presence of two Cra-binding sequences on the gapA promoter, Cra box-1 on F1 and Cra box-2 on F2 fragment.
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To examine the influence of the two Cra boxes on the gapA transcription, we focused detailed analysis on transcription initiation from P1 (located downstream of Cra-box 1) and P1'/P2' promoters (located downstream of Cra-box 2) (see Fig. 4A for the location), in both wild-type E. coli and a mutant lacking Cra. RNA was prepared from exponentially growing cells in LB medium, and subjected to S1 mapping. Both P1 and P1'/P2' transcripts were detected in the wild-type E. coli, but in the cra mutant, significant elevation was observed only for P1 transcript (Fig. 4C). Then we concluded that the Cra-box 1-bound Cra represses transcription initiation from the P1 promoter (Fig. 4A,C).
In order to confirm transcription regulation of the gapA gene by Cra, the activity in vivo of the gapA promoters was examined using the newly developed TFP (two-fluorescent protein) vector (Shimada et al. 2004). Both the full-length F12 (including all the gapA promoters) and the downstream half F1 (without the P1'/P2' promoters) fragments were inserted into TFP vector so as to adjust the gapA initiation codon to that of GFP. In the wild-type E. coli, the GFP activity relative to the RFP, which is under the control of internal reference promoter, was very low for F1 but markedly increased in the mutant lacking Cra (Fig. 4D). When F12 fragment was used, GFP expression was detected even in the wild-type and increased in the cra mutant. The level of GFP increase in the cra mutant was as high as that observed with F1, indicating that the derepression effect is arisen from the P1–P5 promoters (see Fig. 4A for promoter organization). The result indicates that the newly identified upstream promoters P1'/P2' contribute, to certain extent, the expression of gapA at least under the repression conditions. The promoter assay confirmed that gapA transcription from the P1–P5 promoters is repressed by Cra-box 1-bound Cra protein, but the role of Cra-box 2 remains unsolved.
Transcription regulation of the eno gene by Cra
Previously, the eno gene was cloned and its expression was measured by Klein et al. (1996). For search of the Cra-binding site on the eno gene, three species of the promoter fragment were prepared, i.e. a 989 bp-long F12 fragment that covers the entire 5' flanking sequence of eno starting from ATG, 500 bp-long F1 fragment (downstream half of F12; –1 to –500 bp) and 394 bp-long F2 (upstream half of F12; –596 to –989 bp) (Fig. 5A). By gel shift assay, the formation of Cra complex was detected for both F12 and F2 fragments but not for F1 (Fig. 5B), indicating that the Cra-binding site(s) is located between –596 and –989 bp from its translation initiation site. After sequence analysis, the Cra-box was identified in two regions (the conserved T residue at –945 and –748, respectively) within this F2 fragment. These Cra boxes are located within the C-terminal portion of the upstream gene, pyrG encoding CTP synthase.
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To confirm transcription repression of the eno gene by Cra, both F1 and F12 fragments were inserted into the promoter assay TFP vector and measured the GFP expression level relative to RFP level. With the full-length F12 fragment, the GFP level increased in the cra mutant (Fig. 5D). We then concluded that the Cra boxes within F2 region contribute to the repression of ene gene transcription from P1, P2 or P3 promoters. Even though no promoter was detected in F1 region by S1 mapping, F1 supported GFP expression in wild-type E. coli but F1-drived GFP expression decreased in the cra mutant. One possible explanation is that an as yet unidentified promoter in F1 requires Cra for activation.
Transcription regulation of the yahA and hypF genes by Cra
Transcription in vitro of the gapA and eno genes was repressed by Cra in dose-dependent ways (data not shown). Under the same assay conditions, we analyzed transcription regulation of two newly identified genes, yahA and hypF, with unknown functions. By in vitro transcription assay, we detected two promoters (P1 and P2) for the yahA gene (Fig. 6A). In the presence of Cra, the level of P1 RNA decreased in Cra dose-dependent way (up to 40 pmol Cra), while P2 RNA level stays almost constant in the presence and absence of Cra. In the case of gapA and eno transcription in vitro, transcription was almost completely repressed by the addition of 5 pmol Cra (data not shown). The 220 bp-long SELEX fragment isolated in our screening corresponds to the sequence between –332 and –551 with respect to the translation initiation codon (Fig. 6A). Even though a single Cra box within this fragment overlaps with the transcription initiation site of P2 promoter, marked repression takes place for P1.
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Discussion |
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After checking various conditions for SELEX screening using a number of E. coli transcription factors, we found a number of factors influence the successful SELEX screening, including the mixing ratio of DNA and proteins, the number of SELEX cycles, the presence or absence of positive (in the case of activators) or negative (in the case of repressors) effectors affecting the DNA-protein interaction, and the reaction conditions such as solvent compositions, temperature and reaction time (K. Hirao, T. Shimada, K. Yamamoto, N. Fujita and A. Ishihama, unpublished observation).
A total of 13 promoters are known to be under the control of Cra, of which 6 species were identified in our collection of SELEX fragments. All six known SELEX fragments (fruB, epd, adhE, ptsH, nirB, pykF) are the group of genes, which are repressed by Cra. In contrast, all the promoters that are activated by Cra were not selected in our SELEX, because the affinity of Cra to this group promoters is weak under the conditions employed (in the absence of effectors). Among seven promoters that were hitherto identified to be under the control of Cra but not selected by this SELEX, two (pfkA and edd) belong to the promoter group that is repressed by Cra. The affinity of these two promoters to Cra is, however, weaker than those that were selected by SELEX (see Fig. 2). These results altogether indicate that the SELEX screening conditions employed herewith, i.e. repeated screening, allowed to select the promoters with tight binding activity of the Cra protein.
Using the improved method of genomic SELEX, we identified in this study at least four new members of the Cra regulon, including two genes with the known functions. Both the gapA (glyceraldehyde 3-phosphate dehydrogenase) and eno (enolase) promoters showed high affinity to Cra in the absence of inducers (or effectors) as in the case of repression-type gene promoters (see Fig. 2). In agreement with the prediction of metabolic switching by Cra, both enzymes are involved in the equilibrium reactions in the pathways of glycolysis and gluconeogenesis (Harris & Waters 1976; Cunningham et al. 1997).
D-glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) is a key enzyme of glucose metabolism. It plays a crucial role in catabolic and anabolic carbohydrate metabolism, catalyzing the reversible oxidation of D-glyceraldehyde-3-phosphate into 1,3-diphosphoglycerate. In most bacteria, the gap gene encoding GAPDH is organized upstream of the pgk gene encoding phosphoglycerate kinase in a cluster of genes encoding other glycolytic enzymes. However, two gap genes exist in E. coli. Even though the gapB gene is located upstream of the pgk gene, detectable GAPDH activity has not been detected for its gene product (Zhao et al. 1995; Boshi-Muller et al. 1997). Instead, GapB was shown to have non-phosphorylating erythrose 4-phosphate dehydrogenase (E4PDH) activity. The gapA gene codes for a protein that is more similar to eukaryotic than eubacterial GAPDHs (Branlant & Branlant 1985). Expression level of GapA is as high as other glycolytic enzymes while that of GapB is expressed at a very low level (Zhao et al. 1995; Boshi-Muller et al. 1997).
Previously, Charpentier & Branlant (1994) identified four in vivo promoters, of which gapA P2 is recognized by RpoH sigma and gapA P3 is subject to catabolite repression by CRP. Moreover we identified in this study that gapA P1 is repressed by Cra, which binds to the promoter-proximal Cra-box 1. In reflecting the physiological role of GapA as a real GAPDH, the promoter organization is very unique, being controlled by two global regulators, CRP and Cra, for a large set of genes for carbohydrate metabolism. In good agreement with the sophisticated regulation system, these gapA promoters are activated differentially in response to variation in the culture conditions (Charpentier & Branlant 1994). In addition to these four promoters, we identified two minor promoters, P1' and P2', and the promoter-distal Cra-box 2. Since transcription start sites of the P1' and P2' promoters are located at –524/525 and –530 bp, respectively, from the initiation codon, it is not clear yet whether these upstream promoters play regulatory roles in vivo for gapA expression.
Here we also found that Cra-box exists in the eno gene promoter region. The reversible conversion of 2-phosphoglycerate to phosphoenolpyruvate by the enzyme enolase is on the major pathway for the synthesis of phosphoenolpyruvate in glycolysis. Interestingly, enolase is also a component of RNA degradosome (Carpousis 2002) and plays a crucial role in the rapid decay of PtsG glucose transporter mRNA (Morita et al. 2004). Up to now, however, transcription regulation of the eno gene has not been studied. Here we identified three in vivo promoters by S1 mapping, and all three promoters appeared to be derepressed in the absence of Cra protein (see Fig. 5). In addition to the two genes, gapA and eno, with known functions, we identified, by genomic SELEX, two new Cra-target genes, yahA and hypF, with unidentified functions. In vitro transcription assays indicated repression of these two genes by Cra (see Fig. 6). The repression level of in vitro transcription, however, differed among gapA, eno, yahA and hypF, reflecting not only the affinity of each Cra box to Cra protein but also the location of Cra box relative to the respective promoters.
Here we demonstrated that between the genes under the control of Cra, there is a hierarchy in the affinity to Cra (see Fig. 2). Generally the genes, which are repressed by Cra, have higher affinity to Cra than those that are activated by Cra. In the absence of inducers, Cra should first bind to the promoters of repression-group genes. Once the bound Cra on these group promoters are dissociated by inducers, the inducer-bound Cra might be able to associate with the activation-type promoters. The genes carrying the repression-type promoters are mostly involved in the metabolic pathway of glycolysis while the genes with the activation-type promoters are involved in gluconeogenesis. The molecular switching of Cra distribution is a critical factor for metabolic switching between glycolysis and gluconeogenesis.
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Experimental procedures |
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E. coli KP7600 (W3110 lacI lacZ 
galK2 galT22) was used as wild-type strain (Shimada et al. 2004). Starting from KP7600 strain, an cra disruptant strain JD20323 was constructed by a transposon insertion method (Makinoshima et al. 2003). The absence of Cra protein was confirmed by immunoblotting of whole cell lysates. Cells were grown at 37 °C under aeration in Luria-broth (LB) medium. Overnight culture in LB medium was diluted 1000-fold into fresh LB, and the incubation was carried out at 37 °C with shaking at a constant rate (150 r.p.m.). Cell growth was monitored by measuring the turbidity at 600 nm.
Purification of Cra protein
For construction of plasmids pCra for Cra-expression, a DNA fragment corresponding to the Cra-coding region was amplified by PCR using E. coli W3110 genome DNA as a template and a pair of primers, and after digestion with NdeI and NotI, cloned into pET21a(+) (Novagen) at the corresponding sites. The plasmid construct was confirmed by DNA sequencing. For protein expression, the pCra plasmid was transformed into E. coli BL21(DE3). Transformants were grown in 200 mL of LB broth and at the cell density of 0.6 OD600nm, IPTG was added at the final concentration of 1 mM. After 3 h, cells were harvested by centrifugation, washed with a lysis buffer (50 mM Tris-HCl, pH 8.0 at 4 °C, 100 mM NaCl), and then stored at –80 °C until use.
For protein purification, frozen cells were suspended in 3 mL of lysis buffer containing 100 mM PMSF. After adding 80 µL of lysozyme (10 mg/mL), the cell suspension was stored on ice for 30 min and then lyzed by sonication. After centrifugation at 15 000 r.p.m. for 20 min at 4 °C, the resulting supernatant was mixed with 2 mL of 50% Ni-NTA agarose solution (Qiagen) and loaded onto a column. After washing with 10 mL of lysis buffer, the column was washed with 10 mL of washing buffer (50 mM Tris-HCl, pH 8.0 at 4 °C, 100 mM NaCl). Proteins were then eluted with 2 mL of an elution buffer (200 mM imidazole, 50 mM Tris-HCl, pH 8.0 at 4 °C, 100 mM NaCl), and dialyzed against a storage buffer (50 mM Tris-HCl, pH 7.6, 200 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, and 50% glycerol), and analyzed on SDS-PAGE.
SELEX search for Cra-binding sequences
Genome DNA of E. coli W3110 was sonicated to generate fragments of 200–300 bp in length (for details see N. Fujita and S. Endo, unpublished observation). The E. coli DNA library was constructed after cloning of these 100–300 bp DNA fragments into plasmid pBR322 at EcoRV site. A collection of these 100–300 bp DNA fragments could be regenerated by PCR-amplification using the E. coli DNA library plasmids as templates and a set of primers, primer-1 (5'-CTTGGTTATGCCGGTACTGC-3') and primer-2 (5'-GCGATGCTGTCGGAATGGAC-3'), which hybridize with the pBR322 vector at EcoRV junctions. PCR products thus generated were purified by PAGE.
For the genomic SELEX screening of Cra-binding sequences, 5 pmol of DNA fragments and 10 pmol His-tagged Cra were mixed in a binding buffer (10 mM Tri-HCl, pH 7.8 at 4 °C, 3 mM Mgacetate, 150 mM NaCl, BSA 1.25 µg/mL) and incubated for 30 min at 37 °C. The mixture was applied onto Ni-NTA column and after washing unbound DNA with the binding buffer containing 10 mM imidazole, DNA-Cra complexes were eluted with an elution buffer containing 100 mM imidazole. DNA fragments recovered from the complexes were ligated into pBR322 and PCR-amplified as above. If necessary, this SELEX cycle was repeated several times. For sequencing of Cra-bound DNA fragments, PCR products were cloned into pT7 Blue-T vector (Novgen) and transformed into E. coli DH5
. Sequencing was carried out using T7-primer (5'-TAATACGACTCACTATAGGG-3') with ABI DNA sequencer.
S1 nuclease protection assay
S1 nuclease protection assay was carried out as previously described (Yamamoto et al. 2002). Mixtures of the 32P-end-labeled probe (104 cpm) and total RNA (100 µg) were incubated for 10 min at 75 °C for denaturation, and then incubated at 37 °C overnight for hybridization. After digestion with S1 nuclease (TaKaRa) at 37 °C for 10 min, undigested products were extracted with phenol, precipitated with ethanol, and analyzed by electrophoresis on polyacrylamide gels containing 8 M urea. The intensity of undigested probe bands on gels was measured with BAS1000 (Fuji).
Gel mobility shift assay
DNA probes were generated by PCR amplification of promoter region with respective primers and E. coli W3110 genome DNA (80 ng) as the template using Ex-Taq DNA polymerase. The PCR products were purified by polyacrylamide gel electrophoresis, and then terminal-labeled with 10 µCi of [
-32P]ATP (5000 Ci/mmol) by T4 polynucleotide kinase (TaKaRa). After purification by PAGE, the 32P-labeled probe DNA was incubated at 37 °C for 10 min with the purified Cra in 0.01 mL of 50 mM Tris-HCl (pH 7.8), 50 mM NaCl, 3 mM Mg acetate, 0.1 mM EDTA, 0.1 mM dithiothreitol, and 25 µg/mL of BSA. After addition of the DNA dye solution (30% glycerol, 0.25% bromophenol blue, 0.25% xylene cyanol), the mixture was directly subjected to 6% polyacrylamide gel electrophoresis.
DNase I footprinting
DNase I footprinting was carried out as previously described (Yamamoto et al. 2002). In brief, the test protein was incubated with a 32P-labeled DNA fragment in transcription buffer [50 mM Tris-HCl (pH 7.8 at 37 °C), 50 mM NaCl, 3 mM magnesium acetate, 0.1 mM EDTA, 0.1 mM DTT and 25 mg/mL BSA] at 37 °C for 10 min. DNA digestion was initiated by the addition of 5 ng DNase I (Takara). After incubation for 30 s at 25 °C, the reaction was terminated by adding 45 µL of the stop solution. DNA was precipitated with ethanol, dissolved in formamide-dye solution, and analyzed by 6% PAGE in the presence of 8 M urea.
In vitro transcription assay
In vitro transcription assay was performed as previously described (Kajitani & Ishihama 1984; Igarashi & Ishihama 1991) using truncated DNA templates with Cra-binding sequences, which were PCR-generated, and in the presence or absence of purified Cra protein. Transcripts were extracted with phenol, precipitated with ethanol, and analyzed by electrophoresis on polyacrylamide gels containing 8 M urea. The intensity of undigested probe bands on gels was measured with BAS1000 (Fuji Film).
Measurement of in vivo promoter activity
Promoter activity in vivo was measured using the newly developed TFP (two-fluorescent protein) vector (Shimada et al. 2004). Briefly each test promoter of 300–500 bp upstream from the initiation codon is inserted into TFP vector so as to adjust the initiation codon of GFP (green fluorescent protein). The expression level of reporter GFP was quantitated by measuring the ratio of GFP to RFP (red fluorescent protein), which is expressed under the control of a reference promoter on the same vector. For measurement of the fluorescent intensity of RFP or GFP expressed in E. coli, cells grown in LB medium for various times were harvested by centrifugation, resuspended in PBS(–), and diluted with PBS(–) to give approximately the same cell density of 0.6 A600nm for all the samples. For measurement of bulk fluorescence, aliquots of 0.3 mL cell suspension were applied into 0.4 x 96 flat-bottom wells, and the fluorescence was measured with a FL600 Bio-Tek microplate reader (Bio-Tek Instruments, USA). Since LB medium contains yet unidentified natural substances expressing fluorescence albeit at a low level, the background was subtracted for each sample.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: aishiham{at}nibs.or.jp
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References |
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Received: 21 February 2005
Accepted: 20 June 2005
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) show higher affinity than activation-type promoters (
).
