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pR promoter by Escherichia coli SeqA protein requires downstream GATC sequences and involves late stages of transcription initiation
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1
grzyn1grzyn2
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1 Department of Molecular Biology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland
2 Laboratory of Molecular Biology (affiliated with University of Gdansk), Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Kladki 24, 80-822 Gdansk, Poland
Correspondence
Agnieszka Szalewska-Paasz
szalewsk{at}biotech.univ.gda.pl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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promoters pI, paQ and pR. While SeqA-mediated stimulation of pI and paQ occurs by facilitating functions of another transcription activator protein, cII, a mechanism for stimulation of pR remains largely unknown. Here, it has been demonstrated that two GATC sequences, located 82 and 105 bp downstream of the pR transcription start site, are necessary for this stimulation both in vivo and in vitro. SeqA-mediated activation of pR was as effective on a linear DNA template as on a supercoiled one, indicating that alterations in DNA topology are not likely to facilitate the SeqA effect. In vitro transcription analysis demonstrated that the most important regulatory effect of SeqA in pR transcription occurs after open complex formation, namely during promoter clearance. SeqA did not influence the appearance and level of abortive transcripts or the pausing during transcription elongation. Interestingly, SeqA is one of few known prokaryotic transcription factors which bind downstream of the regulated promoter and still act as transcription activators. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; von Freiesleben et al., 1994; Slater et al., 1995). This regulation appears to be mediated by binding of SeqA to hemimethylated GATC sequences (Bach & Skarstad, 2005). Nevertheless, it appears that the cellular function of SeqA is not as simple as supposed shortly after its discovery. Subsequent studies (exemplified briefly below) have revealed many additional activities of SeqA which shed new light on its roles in cell biology.
SeqA is an inhibitor of the onset of E. coli chromosome replication in vivo (Boye et al., 1996) and in the presence of high concentrations of the replication initiator protein DnaA in vitro (Wold et al., 1998); however, it may stimulate DNA synthesis at low DnaA concentrations in vitro (Wold et al., 1998). Studies on DNA supercoiling and the localization of SeqA in cells suggested that this protein affects organization of the nucleoid (Hiraga et al., 1998; Weitao et al., 1999). It has been observed that in the absence of SeqA, negative supercoiling of DNA increases, and purified SeqA generates positive DNA supercoils in vitro, strongly suggesting that this protein may take part in the organization of the chromosome in vivo (Klungsoyr & Skarstad, 2004; Odsbu et al., 2005). Results of experiments in which SeqA was overproduced suggested that the function of this protein in the regulation of replication initiation is linked to chromosome segregation and cell division (Bach et al., 2003). SeqA could be a global cellular regulator, involved in the formation of so-called hyperstructures (Norris et al., 2002; Molina & Skarstad, 2004). Moreover, there is evidence indicating that SeqA is also involved in the protein degradation process, as in the absence of functional SeqA, the mutant DnaA204 protein has been shown to be stabilized (Torheim et al., 2000; Somiska et al., 2003c).
Perhaps an unexpected finding was discovery that permeability, and possibly other properties of E. coli cellular membranes, are significantly altered in a seqA mutant relative to wild-type bacteria (Wgrzyn et al., 1999). One possible explanation for this phenomenon could be SeqA-mediated regulation of the expression of certain genes. In fact, subsequent studies have revealed that SeqA is also a specific transcription factor. It can stimulate activities of certain promoters both in vivo and in vitro, including pI, paQ and pR of bacteriophage , while other promoters, like pE and pL, are not influenced (Somiska et al., 2001, 2003a, b).
An intriguing question is how can SeqA, a regulator of DNA replication, control transcription initiation at certain promoters. Lobner-Olsen et al. (2003) performed a microarray analysis to show that in a seqA mutant the levels of some transcripts were significantly decreased while certain transcripts were evidently more abundant relative to wild-type bacteria. However, they failed to find any correlation between the presence of GATC motifs (SeqA-binding sites) in promoter sequences and transcription activity and suggested that SeqA-mediated regulation of transcription may be indirect. However, subsequent studies have indicated that when larger DNA fragments, encompassing positions –250 to +250 relative to the transcription start site, were analysed, some common features of GATC distribution near the promoters activated by SeqA could be suggested (Strzelczyk et al., 2003). In such cases, two or more GATC motifs are often located downstream of a stimulated promoter. This also applies to promoters whose activities have to date been demonstrated to be stimulated by SeqA in vitro ( pI, paQ and pR) and thus for which a direct stimulatory mechanism can be assumed (Strzelczyk et al., 2003).
In vitro transcription studies indicated that SeqA stimulates transcription from pI and paQ promoters by facilitating the function of another activator, the cII protein (Somiska et al., 2003b). The cII gene product is a positive regulator of these promoters and it has been proposed that in the presence of SeqA it may bind to these promoter sequences more efficiently. This is in contrast to pE, another cII-dependent promoter, which has been demonstrated to be insensitive to SeqA. Interestingly, DNA templates used in in vitro transcription assays from pE did contain GATC sequences upstream, but not downstream of the promoter (Somiska et al., 2003b).
Contrary to pI and paQ, a mechanism for stimulation of pR activity by SeqA has remained unclear. Although pR is also stimulated by another transcription activator, DnaA (Szalewska-Paasz et al., 1998; Glinkowska et al., 2003), it has been demonstrated that there is competition between SeqA and DnaA at pR rather than cooperation (Somiska et al., 2003a). Therefore, a mechanism analogous to that observed for pI and paQ is not possible for pR. On the other hand, since SeqA is a global regulator of various crucial cellular processes (see above), including regulation of gene expression, it would appear to be important to elucidate a mechanism for the stimulation of activity of one of these model promoters, pR, by this protein. Therefore, the aim of this work was to learn about the mode of SeqA-mediated activation of the pR promoter. Our in vivo and in vitro studies revealed that two GATC sequences located downstream of the pR transcription start site are crucial for this activation, which is independent of DNA topology and can occur after open complex formation.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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(argF-lac)U169 (his-gnd) thi rpsL150 glt0 flbB5301 relA1 deoC1 rbsR], described by Thomas & Glass (1991) and referred to as seqA+ (or wild-type, WT), and its seqA : : Tn10 derivative, BM761 (Somiska et al., 2001), were used in in vivo experiments. The MG1655 wild-type strain and its dam13 : : Tn9 derivative (Somiska et al., 2001) were employed to obtain methylated and unmethylated plasmid DNAs, respectively.
Plasmids.
Plasmid pTAC3734 (Brondsted & Atlung, 1994), bearing a promoterless lacZ reporter gene, was used for construction of pR-lacZ fusions. The wild-type pR promoter region (207 bp, from –73 to +135), bearing two GATC sequences located at positions +82 and +105 relative to the transcription start site (named box A and box B, respectively, in this report) was amplified by PCR using primers pR-Sma and pR-Hind2, which introduced SmaI and HindIII restriction sites, respectively, at the ends of the product (Table 1). This DNA fragment was inserted into the corresponding sites of pTAC3734. The GATC motifs were then changed for CATC by PCR-mediated site-directed mutagenesis, using primers 1mutA1 and 1mutA2 (for mutating box A) and 1mutB1 and 1mutB2 (for mutating box B). Thus, a series of plasmids bearing a wild-type pR-lacZ fusion or its derivatives bearing either mutated box A or box B or both, was obtained.
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) was used as a vector. This plasmid bears the sequences of strong terminators located downstream of the cloning sites. The pR promoter fragments (wild-type and those harbouring mutations) were transferred from the pTAC3734-derived plasmids into pTE103 by insertion of the SmaI–HindIII fragments bearing the pR region to corresponding sites of the recipient. Thus, a series of plasmids analogous to that described above, was obtained.
All molecular cloning procedures were performed according to Sambrook & Russell (2001). All constructs were verified by DNA sequencing.
Proteins.
SeqA protein, purified as described by Skarstad et al. (2000) was provided by Dr Kirsten Skarstad (Institute for Cancer Research, Oslo, Norway). The 
70-containing RNA polymerase holoenzyme was purchased from Epicenter Technologies.
Measurement of 
-galactosidase activity in E. coli cells.
-Galactosidase activity in bacterial cells was measured according to a previously described method (Miller, 1972). Since multicopy gene fusions (located on plasmids) were used, the results were normalized per amount of plasmid DNA in cells; plasmid copy number in E. coli was estimated by isolation of plasmid DNA from a known number of cells, linearization with restriction endonuclease, separation during agarose gel electrophoresis, staining with ethidium bromide and densitometric analysis of the bands relative to known amounts of plasmid DNA separated on the same gel, as described by Wgrzyn et al. (1996).
In vitro transcription assay.
Either linear or supercoiled DNA templates were used for the reactions. Unmethylated linear templates were obtained by PCR using pTAC3734-derived plasmids and pR-Sma and pR-Hind2 primers (Table 1
). Fully methylated linear templates were obtained by treatment of the PCR products (2 µg) with 10 units Dam methylase (New England BioLabs) for 1 h at 37 °C. Methylated and unmethylated supercoiled templates were obtained by isolation of pTE103-derived plasmids from dam+ or dam13 : : Tn9 bacteria, respectively, and purification of supercoiled plasmid DNA by ultracentrifugation in a CsCl-ethidium bromide density gradient (Sambrook & Russell, 2001).
The in vitro multi-round transcription reactions were performed in a total volume of 25 µl in buffer M (20 mM HEPES, pH 8.0, 5 mM magnesium acetate, 4 mM DTT, 1 mM EDTA, 1 mM ATP, 5 mg BSA ml–1, 0.2 % Triton X-100, 5 % glycerol). Template DNA (12.5 nM) and the indicated amounts of the SeqA protein were used. The binding reaction of SeqA was carried out for 10 min at 37 °C, then RNA polymerase was added (to a final concentration of 50 nM) and incubation was continued for the next 10 min. After the addition of nucleotides {CTP and GTP up to a final concentration of 150 µM, ATP up to 1 mM, UTP up to 15 µM and [
-32P]UTP up to 1 µCi ml–1 (3.7x104 Bq)}, the samples were incubated at 37 °C for 12 min. Following the addition of heparin (100 µg ml–1) and further incubation for 5 min, the reaction was halted by addition of 5 µl stop buffer (150 mM EDTA, 1.05 M NaCl, 7 M urea, 10 % glycerol, 0.0375 % xylene cyanol, 0.0375 % bromophenol blue). The samples were separated by electrophoresis in a 4.5 % polyacrylamide gel containing 7 M urea in the TBE buffer (Sambrook & Russell, 2001) at 350 V. The gel was dried and RNA bands were visualized and quantified using the PhosphorImager system (Bio-Rad).
For single-round transcription, heparin was added to the NTP mixture to a final concentration of 100 µg ml–1.
Order of addition in in vitro transcription assays.
The wild-type supercoiled DNA template was used in all these assays. The buffer, temperature and nucleotide concentration were as described for the assay of in vitro multi-round transcription. Pre-incubation of the template with either SeqA (final concn 1 µM) or RNA polymerase (50 nM) for 10 min was followed by addition of the second protein (alternatively, both proteins were pre-incubated in the absence of DNA). The transcription reaction was initiated by addition of nucleotides and heparin (100 µg ml–1) at various times (including time 0) after the addition of the second protein. The mixture was incubated for 12 min and then the reaction was stopped, and analysis of samples was performed as described for the assay of in vitro multi-round transcription.
Estimation of promoter escape efficiency.
Reactions were performed in a total volume of 80 µl in M buffer (see above) using the wild-type supercoiled template. The template was incubated with RNA polymerase (50 nM) for 20 min at 37 °C, and then heparin was added to a final concentration of 100 µg ml–1, either together with SeqA (1 µM) or without this protein. The reaction was started by the addition of a nucleotide mix (see description of in vitro multi-round transcription assay). Samples (12 µl each) were withdrawn at 0, 0.5, 1, 2 and 4 min after the onset of the reaction and the reaction was quenched with stop buffer. Samples were separated by electrophoresis and analysed as described for the assay of in vitro multi-round transcription.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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omiska et al., 2001, 2003a; Strzelczyk et al., 2003), we tested the dependence of SeqA-mediated stimulation of pR on the presence and position of GATC sequences. Previous studies indicated that maximal activation by SeqA could be achieved when using DNA templates bearing only proximal GATC motifs located downstream of pR (Somiska et al., 2001, 2003a). Therefore, a series of pR-lacZ fusions was constructed, containing the pR promoter region with two GATC sequences (named A and B) located at positions +85 and +102 (with respect to the first nucleotide of the GATC box), which were either wild-type or mutated. In the latter cases, the G was replaced with C in box A, box B or both. This mutation abolished SeqA binding to the specific sequences (Fujikawa et al., 2004).
In vivo pR promoter activity (assessed as -galactosidase reporter activity in cells containing the fusions) indicated that scrambling of one of the GATC motifs (either A or B) or both resulted in significant impairment of SeqA-mediated stimulation of pR transcription. The effects were more dramatic in the case of disruption of box A and both boxes (Fig. 1); when only box A was present, SeqA retained some stimulatory effect on pR. These results suggest that both GATC motifs, that serve as SeqA-binding sites, are required for pR stimulation.
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omiska et al., 2001). In these experiments, we used either fully methylated or unmethylated DNA templates. It was demonstrated previously that SeqA can activate pR located on both hemimethylated and fully methylated templates (with equal efficiency), but not on unmethylated DNA (Somiska et al., 2001). We found that the maximal level of pR activation by SeqA was about two- to threefold when activator protein was present at optimal concentration (1 µM SeqA for 50 nM RNA polymerase and 12.5 nM template) and when a wild-type fully methylated DNA template was used, which was in agreement with previous observations (Somiska et al., 2001). The in vitro activation was directly dependent on the presence of intact GATC sequences (Fig. 2). Mutations in these motifs notably impaired the activation by SeqA (box B) or abolished it completely (box A or both). As expected, the activation was strictly dependent on methylation of the template: when unmethylated template was used, no significant activation was observed, irrespective of the status of GATC motifs (Fig. 2). A similar effect was observed for single-round transcription assays (data not shown).
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SeqA-mediated stimulation of pR is independent of DNA topology
Since SeqA can affect DNA topology (see Introduction) and the activity of various promoters may depend on template supercoiling (for review, see Muskhelishvili & Travers, 2003), we constructed a supercoiled DNA template for in vitro transcription assays. This was achieved by introducing the analogous DNA fragment that was used in previous assays (bearing the pR region with wild-type GATC sequences and with mutated box A, box B or both) into a pTE103 plasmid vector (Elliot & Geiduschek, 1984). This vector bears strong termination signals downstream of the cloning site, and thus can be employed in studies of promoter activity in vitro using a supercoiled template. The constructed plasmids were introduced into a dam+ host and an otherwise isogenic dam mutant, and either methylated or unmethylated templates were obtained following isolation of plasmids from these hosts, respectively.
We found that in the in vitro multi-round transcription experiments the efficiency of SeqA-mediated activation of pR transcription on the methylated template bearing the wild-type promoter region was comparable to that observed in reactions with a linear template (compare Fig. 3 and Fig. 2, WT). Therefore, we conclude that template supercoiling has little or no influence on stimulation of pR by SeqA. Similar to experiments with linear templates, this stimulation was abolished in the absence of methylation and was dependent on the presence of intact GATC motifs, confirming that SeqA exerts its stimulatory role through binding to at least one of two GATC motifs on supercoiled templates (Fig. 3).
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In the in vitro experiments described previously, SeqA was usually preincubated with the DNA template, allowing its binding to the appropriate GATC sequences and thus we observed transcription from pR after the set time of the reaction. In subsequent experiments we measured time-dependent accumulation of the full-length transcript after addition of RNA polymerase. Briefly, after pre-incubation of the template with SeqA (or with a buffer in the control experiments) for 10 min, and addition of RNA polymerase holoenzyme, aliquots were withdrawn at various times (including time 0) and the reaction was started with the addition of NTPs, together with heparin, and stopped after 12.5 min. The reaction products were separated electrophoretically and the amounts of full-length products were analysed. In these types of experiment we observed about twofold higher amounts of pR-derived transcripts in the presence of SeqA than in its absence (in agreement with the level of activation observed previously); however, in both cases, increased amounts of transcripts appeared after similar times of incubation of the template with RNA polymerase (Fig. 4). Therefore, we concluded that SeqA did not shorten the time necessary for achieving maximal transcription output.
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A regulatory protein can also act at the stage of promoter escape during transcription. To estimate pR promoter escape efficiency, RNA polymerase was allowed to bind the template (20 min preincubation) and then heparin was added (to prevent reinitiation) together with SeqA (or buffer in control experiments). The reaction was started by the addition of NTPs and samples were withdrawn at various time points after the onset of the reaction. We found that although SeqA did not shorten the time from the start of the reaction to production of the first full-length transcripts, the kinetics of the process appeared to be faster and formation of transcription products was significantly more efficient in the presence of SeqA protein (Fig. 5). Note that SeqA was added after the stages of RNA polymerase binding to the template and open complex formation.
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), was studied in an in vitro transcription assay designed as for promoter escape, where the samples were withdrawn at times shortly after the start of the reaction and the products were analysed on 25 % sequencing gels. No effect of SeqA on the appearance of abortive transcripts was observed (data now shown). Moreover, this experimental design allowed us to determine the influence of SeqA on pausing during transcription from pR. The analysis of transcript products appearing during transcription revealed that SeqA does not alter the presence or relative strength of pause sites (data not shown). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), certain promoters of bacteriophage (pI, paQ and pR) have also been shown to be stimulated by SeqA (Somiska et al., 2001, 2003a, b) with significant effects on the development of (Wgrzyn et al., 1999; Somiska et al., 2001, 2003a, b; Wgrzyn, 2006).
The function of SeqA in the regulation of replication is dependent on a hemimethylated state of DNA, while in transcription activation, SeqA is able to activate pR when DNA is fully methylated (Figs 2 and 3; Somiska et al., 2001, 2003a). SeqA exerts its effect when bound to specific GATC sequences. Two such motifs, located at 82 and 105 bp downstream of the transcription start site, are crucial for SeqA-mediated stimulation of pR both in vivo and in vitro (Figs 1–3). Specific binding of SeqA to this DNA region has been demonstrated previously (Somiska et al., 2001, 2003a). The optimal SeqA-mediated stimulation of pR activity was achieved when both of the GATC sequences were present and intact. However, box A, proximal to the promoter, seems to be more important for this effect, possibly indicating that the action of SeqA is facilitated by the site located closer to the promoter.
The downstream location of the activator-binding site raises questions about the possibility of a roadblock for RNA polymerase. For DnaA, acting from the +18 position in pR transcription, binding has been shown to be relatively weak (Szalewska-Paasz et al., 1998; Glinkowska et al., 2003). Moreover, it has also been shown that an excess of both DnaA (Szalewska-Paasz et al., 1998) and SeqA (this work, data not shown) inhibit pR activity.
Taking into the account the distance apart and sizes of the proteins, direct interaction of SeqA with RNA polymerase is not easy to predict unless a significant DNA bend occurs (not likely due to the short distance), thus indicating a more indirect mode of action. As we demonstrated that SeqA-mediated activation of pR is independent of DNA topology (compare Figs 2 WT and 3) it is unlikely that SeqA acts through facilitating DNA destabilization near the activated promoter, since template supercoiling is crucial in this process (Sheridan et al., 1998).
In vitro analysis determined that the presence of SeqA did not shorten the time necessary for achieving the maximal transcriptional output (Fig. 4). Twice the initial level of SeqA-mediated transcription (Fig. 4, time zero) may indicate the possible action of SeqA on the initial stages. However, this phenomenon may be a result of other steps in the transcription process affected by the presence of SeqA (which would be observed as an increase in transcriptional output). Besides, it is unlikely that SeqA could stimulate transcription by open complex stabilization because for pR this is not a rate-limiting step (McKane & Gussin, 2000). Measurement of the efficiency of promoter escape demonstrated notably more efficient transcription from pR in the presence of SeqA compared to the maximal activation noted in a standard in vitro transcription assay (compare Figs 3 and 5). Hence, we conclude that SeqA stimulates pR promoter activity at the stage(s) following formation of the open complex, possibly by facilitating promoter clearance. However, the precise mechanism by which this protein performs such an action, perhaps not contacting RNA polymerase directly, remains to be elucidated.
In the light of the finding that SeqA exerts its regulatory role in the later stages of transcription, one could assume that the regulation could be exerted by affecting the efficiency of productive elongation. However, the presence of SeqA resulted in no notable differences in the amount or time-dependent pattern of such products. Similarly, we could not see any differences in the appearance of pause sites or pausing efficiency in the experiments with or without SeqA protein. Thus, we suggest that SeqA is unlikely to promote its stimulatory function on pR by influencing abortive transcription or pausing of RNA polymerase. The possibility that SeqA affects transcription termination at tR1, a terminator downstream of pR in the genome, can be ruled out as DNA fragments present in the templates used in this work did not contain tR1, but stimulatory effects on pR-initiated transcription were still evident.
In summary, we have demonstrated that SeqA-mediated stimulation of pR transcription is independent of DNA supercoiling, requires two GATC motifs located downstream of the transcription start site and is dependent on template methylation. The major regulatory effect of SeqA in pR transcription occurs after the formation of open complexes in transcription initiation, e.g. at promoter clearance. Our findings place SeqA in the group of prokaryotic positive regulators of transcription which bind downstream of the transcription start site. Such a feature, found frequently in eukaryotic promoters, is very rare in prokaryotes and is exemplified only in specific cases of DnaA (Szalewska-Paasz et al., 1998; Glinkowska et al., 2003), Ler (Sperandio et al., 2000) and some activators from the AraC family (Rns, CfaR, VirF, AggR and CsvR) (Martin & Rosner, 2001; Munson et al., 2001).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 2 May 2006;
revised 4 July 2006;
accepted 6 July 2006.
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) or unmethylated (
), containing wild-type GATC motifs (WT) or mutant derivatives, bearing G to C change(s) in the GATC box A, box B or both boxes, with the indicated amounts of the SeqA protein used in the reaction mixture. Following 10 min pre-incubation of SeqA with a template, RNA polymerase was added and incubation was continued for the next 10 min. Then, transcription was started by the addition of the nucleotide mix and the reaction was continued for 12.5 min. The value obtained for each template in an experiment without SeqA was assumed to be 1 and other values are relative to this value. The results are mean values from three experiments (in each case SD was below 10 %).
