From the
Department of Molecular Biology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland, the
Max-Planck-Institute for Molecular Genetics, Ihnestrasse 73, D-14195 Berlin-Dahlem, Germany, and the **Institute of Oceanology, Polish Academy of Sciences, Sw. Wojciecha 5, 81-347 Gdynia, Poland
Received for publication, December 9, 2002 , and in revised form, March 24, 2003.
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ABSTRACT |
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INTRODUCTION |
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DnaA may cause a premature termination of transcription (5), and it can act as either a repressor or activator of transcription initiation. DnaA-mediated transcription termination might be explained simply by binding of this protein to DnaA-binding sites (DnaA boxes) located within a transcribed DNA region and formation of a barrier for RNA polymerase. Similarly, repression of transcription initiation (promoters repressed by DnaA are exemplified by those for dnaA, mioC, rpoH, and uvrB genes; for a review, see Ref. 4) may be ascribed to binding of DnaA to specific sequences near the promoter and inhibition of RNA polymerase binding. However, mechanisms of transcription activation by DnaA seem to be more complicated. DnaA-mediated stimulation of transcription of several E. coli genes (nrd, glpD, fliC, and polA) was reported (4, 6), but the mechanism of this process remains completely unknown.
Bacteriophage pR promoter was also shown to be stimulated by DnaA both in vivo and in vitro (7, 8). This stimulation of pR, which is an immediate early promoter of the phage, seems to play a crucial role in the control of the frequency of DNA replication initiation at ori
(9, 10) and in the regulation of directionality of this replication (1113). This is because transcription from pR gives mRNA for synthesis of
replication proteins, O and P, and also serves as a process called transcriptional activation of ori
, i.e. transcription proceeding into the region of the origin and stimulating initiation of bidirectional
DNA replication. In fact, certain dnaA mutants cannot be transformed by wild-type
plasmids (i.e. plasmids bearing the bacteriophage
replication region, and containing ori
as the only replication start site), and this phenomenon is partially because of impaired transcription from the pR promoter (14).
Genetic analysis suggested that DnaA may contact the subunit of RNA polymerase during activation of the pR promoter, as effects of specific point mutations in the dnaA gene could be suppressed by certain point mutations in the rpoB gene in an allele-specific manner (8). Moreover, it was demonstrated that during activation of pR the DnaA protein binds downstream of the promoter (8). In fact, there are several potential weak DnaA boxes located downstream of pR (Fig. 1), and electron microscopy studies demonstrated that the DnaA protein binds to these sites (15). Such a binding downstream of a promoter is unusual for prokaryotic transcription activators, but it is rather common in eukaryotic systems.
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Despite determination of some basic rules of DnaA-mediated activation of the pR promoter (summarized above), the molecular mechanism of this phenomenon remained unknown. Therefore, the aim of this work was to investigate this mechanism, especially to identify requirements for particular DnaA box(es) and to determine the step(s) of transcription initiation stimulated by the DnaA protein. Moreover, it was demonstrated previously that at high concentrations DnaA represses the pR promoter rather than activates (8). Therefore, here the mechanism of this repression was also investigated.
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EXPERIMENTAL PROCEDURES |
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Construction of Plasmids Bearing Mutated DnaA Box Sequences The mutant plasmids were constructed by site-directed mutagenesis according to Langer et al. (20). A pair of complementary primers bearing the desired mutation and two external primers (flanking the whole region described below) were used in separate PCR to obtain two partially overlapping fragments. Those fragments were subsequently mixed in a hybridization buffer (33 mM Tris acetate, pH 7.9, 10 mM Mg-acetate, 66 mM K-acetate, 0.5 mM DTT),1 heated to 94 °C, and allowed to cool down slowly. In the next step, hybridized fragments were elongated by T4 DNA polymerase (1 unit) in the presence of 200 mM dNTPs, and used for secondary PCR with external primers. Obtained full-length fragments were subsequently cut with appropriate restriction enzymes (NsiI in the case of boxes 1, 2, and 3, and MunI-SspI in the case of boxes 4, 5, and 6) and cloned into the pLamber vector. Primer sequences and changes introduced by mutagenesis are listed in Tables I and II, respectively. All primers used for introducing mutations have their complementary counterparts (sequences not shown).
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ProteinsDnaA protein was purified as described by Schaper and Messer (21). RNA polymerase holoezyme was purchased from Epicentre Technologies (Madison, WI).
Efficiency of TransformationTransformation of bacterial cells with plasmid DNA was performed by the calcium chloride method according to Sambrook et al. (22). 0.1 µg of plasmid DNA was added to an excess of competent cells and, after the transformation procedure, the efficiency of transformation was calculated on the basis of number of colonies formed after overnight incubation on plates with a selective medium.
In Vitro TranscriptionIn vitro transcription experiments were performed as run-off transcription reactions according to Szalewska-Palasz et al. (8). For preparation of linear templates for in vitro transcription, 311-bp DNA fragments ( coordinates 37,939 to 38,250) with wild-type or mutated DnaA boxes were amplified during PCR using primers 16 and 17 (Table I) and appropriate plasmids as templates. Reaction products were separated electrophoretically and quantified using the PhosphorImager system (Bio-Rad).
Abortive in Vitro Transcription AssayThe HindIII-NsiI fragment of the pRM(minus) plasmid (1 nM) was incubated with various concentrations of DnaA protein in M buffer (20 mM HEPES, pH 8.0, 5 mM magnesium acetate, 4 mM DTT, 1 mM EDTA, 1 mM ATP, 5 mg/ml bovine serum albumin, 0.2% Triton X-100, 5% glycerol) for 10 min at 37 °C. Subsequently, 0.5 unit (15 nM) of RNA polymerase was added and incubation was continued for another 15 min. Reaction was started by adding nucleotides (0.5 mM dinucleotide ApU, 50 µM GTP, 5 µM UTP, 2 µCi of [-32P]UTP). After 12 min at 37 °C, the reaction was terminated with a stop solution (7 M urea, 0.1 M EDTA, 0.4% SDS, 40 mM Tris-HCl, pH 8.0, 0.05% bromphenol blue, 0.05% xylene cyanole). Samples were loaded onto 20% polyacrylamide sequencing gel (acrylamide:bisacrylamide ratio 59:1), separated electrophoretically, and bands were visualized by phosphorimaging.
Gel Retardation AssayReactions were performed in the M buffer containing 50 µg/ml poly(dI-dC). 50 ng of a PCR fragment obtained using primers 16 and 17 (Table I) were mixed with DnaA protein (at indicated concentrations), incubated for 10 min at 37 °C, and separated electrophoretically on a 4% polyacrylamide gel (acrylamide:bis-acrylamide 29:1), in 0.5x TBE buffer at 4 °C. Bands were visualized by SYBR Green staining and analyzed using FluoroImager (Amersham Biosciences).
DNase I FootprintingPCR fragments, obtained using 5'-32P-labeled primers 16 and 17 (Table I) and bacteriophage DNA, were used as a template (100 count/s/reaction). The template was incubated with DnaA protein (at indicated concentrations) for 10 min at 37 °C in the binding buffer, composed of 40 mM HEPES, pH 7.6, 50 mM potassium glutamate, 5 mM magnesium acetate, 1 mM ATP, 0.5 mg/ml bovine serum albumin, 5% glycerol, 0.5 mM DTT, 10 mM MgCl2, and 5 mM CaCl2. Then, 1 (30 nM) or 0.2 units (6 nM) of RNA polymerase was added (where indicated) and incubation was carried on for another 15 min. DNase I was added to a final concentration 0.4 milliunits/µl and after 2 min incubation at 37 °C the reaction was quenched with an equal volume of a stop solution (1% SDS, 200 mM NaCl, 20 mM EDTA pH 8.0), extracted with phenol/chloroform (1:1, v/v) and precipitated with 2 volumes of 96% ethanol. Samples were centrifuged, dried, and resuspended in 4 µl of H2O. Then, 4 µl of the loading buffer (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, 0.05% xylene cyanole) was added, and samples were loaded onto a 8% polyacrylamide sequencing gel containing 8 M urea. After separation, the gel was dried and bands were visualized by phosphorimaging.
KMnO4 Footprinting1 µg of pKB2 plasmid DNA was incubated for 20 min at 37 °C in the binding buffer (see DNase I footprinting) with 1 unit of RNA polymerase. Then, heparin was added to a final concentration of 100 µg/ml, and the samples were incubated for another 10 min with various concentrations of DnaA protein (10, 20, 50, 100, and 200 nM). KMnO4 solution was added to a final concentration of 8 mM, and after another 2 min at 37 °C the reaction was quenched with 2-mercaptoethanol. Samples were extracted with phenol and precipitated with 2 volumes of 96% ethanol. Following suspension of the DNA pellet in water, NaOH was added to a final concentration of 10 mM, and the DNA solution was incubated at 80 °C for 2 min, cooled, and primer extension was performed using 5'-32P-labeled primer 18 (10 pmol) (Table I). The reaction mixture, containing 50 mM Tris-HCl, pH 7.2, 10 mM MgSO4,2mM DTT, was incubated at 50 °C for 3 min to allow primer hybridization. Following addition of dNTPs (5 mM each) and 1 unit of the Klenow fragment of DNA polymerase I, the chain elongation was carried out at 50 °C. After 10 min the reactions were quenched with 1/3 volume of the stop solution (4 mM ammonium acetate, 20 mM EDTA). DNA was precipitated and separated electrophoretically on 8% polyacrylamide-urea sequencing gel.
Estimation of Promoter Escape EfficiencyReactions were performed in a total volume of 80 µl, in the M buffer using 1 mM HindIII-NsiI fragment of pRM(minus) plasmid DNA as a template. The template was incubated with RNA polymerase (0.5 units) (30 nM) for 20 min at 37 °C, and then heparin was added to a final concentration of 60 µg/ml, together with DnaA (35 nM). The reaction was started by addition of a nucleotide mixture (100 µM ATP, CTP, and GTP (each), 10 µM UTP, and 10 µCi/reaction [-32P]UTP). Samples (12 µl each) were withdrawn at 0.25, 0.5, 1, 2, 4, and 8 min after the onset of the reaction, and the reaction was quenched with a stop buffer (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, 0.05% xylene cyanole). Simultaneously, identical control reactions without the DnaA protein were always performed. Samples were separated by electrophoresis in a 6% polyacrylamide gel containing 8 M urea. Phosphorimaging and densitometry were performed subsequently using the Bio-Rad phosphorimaging system.
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RESULTS |
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ColE1-like plasmids cannot replicate in E. coli strains deficient in polA gene function (24). Therefore, transformation of the polA1 mutant by the double-origin, -ColE1 hybrid plasmid could be efficient only when ori
was active. In such an experimental system, transformation was effective in the case of all constructs except those with either box 1 or box 2 scrambled (Fig. 2). These results suggest that ori
is inactive in the absence of box 1 or box 2, most probably because of impaired DnaA-mediated stimulation of transcriptional activation of the origin.
Because boxes 1 and 2 seem to be necessary for activation of pR by DnaA, we have changed the orientation of each of these boxes. These changes did not influence the efficiency of transformation of the polA1 host (Fig. 2). Similar results, i.e. efficient transformation, were obtained when box 1 was moved either 5 or 10 bp away from the pR transcription start site (Fig. 2).
Effects of Changes in DnaA Boxes on in Vitro Transcription from the pR PromoterBecause the transformation assay provided only indirect suggestions about the requirement of particular DnaA boxes for DnaA-mediated stimulation of pR, in vitro transcription assays were performed using templates containing different configurations of DnaA boxes, as described in the preceding paragraph.
Efficient activation of pR was observed at relatively low DnaA concentrations, when wild-type template was used (Fig. 3). In the same experiment, repression of the promoter was detected at high DnaA concentrations, in accordance to previously published results (8). However, when either box 1 or box 2 were scrambled, neither activation nor repression of the pR promoter was observed (Fig. 4A). These results demonstrate directly that box 1 and box 2 are necessary for DnaA-mediated regulation of pR activity. DnaA efficiently activated (at low concentrations) and repressed (at high concentrations) the pR promoter in the absence of other DnaA boxes. This holds as well for reactions in which a short template completely devoid of boxes 36 was used (data not shown). When an even shorter template was used, containing only one DnaA box (box 1), we observed no DnaA-mediated activation and repression of pR (data not shown). Therefore, boxes 1 and 2 are both necessary and sufficient for this regulation.
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Efficient stimulation of pR by DnaA was also observed when box 1 or box 2 were inverted (Fig. 4B). However, inversion of box 1 resulted in maximal activation of pR at higher DnaA concentrations relative to the wild-type template, and inversion of box 2 resulted in a lower efficiency of the activation (Fig. 4B).
Increasing the distance between the transcription start site and box 1 (18 bp from site +1 to the center of the box in the wild-type template) by 5 bp (a half of a helical turn) had no significant effect on DnaA-mediated regulation of pR activity (Fig. 4C). However, further increase of this distance by another 5 bp (i.e. 10 bp, about one helical turn, relative to the wild-type position) resulted in a significant decrease in the efficiency of the promoter stimulation (Fig. 4C).
An Ideal Box 1 Decreases the Efficiency of Transcription Activation of pR by DnaAAll the DnaA boxes located between pR and ori have weak affinity to DnaA (8, 15). Therefore, it was interesting to investigate the effects of replacing the wild-type box 1 with the DnaA box consensus sequence ("ideal" DnaA box), known to bind the DnaA protein strongly.
The -ColE1 hybrid plasmid bearing the ideal DnaA box centered at position +18 transformed the polA1 host efficiently (Fig. 2). In in vitro transcription using the DNA template with the ideal box 1, some DnaA-mediated stimulation of transcription was observed (Fig. 4D). However, this stimulation was of low efficiency and occurred at very low DnaA concentrations.
DnaA Affects Abortive Transcription from pR Similarly to Normal TranscriptionWe asked which step of transcription from the pR promoter is affected by DnaA. Because one of the DnaA boxes necessary for DnaA-mediated regulation of this transcription is located 18 bp downstream of pR, it was likely that transcription initiation is regulated by DnaA. According to this prediction, we found that effects of DnaA on abortive transcription from pR are similar to those observed in standard in vitro transcription reactions (Fig. 5). Therefore, we conclude that DnaA affects initiation of transcription at pR.
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DnaA Stimulates Binding of RNA Polymerase to the pR Promoter RegionTo investigate the mechanism of DnaA-mediated activation of transcription initiation at the pR promoter, each stage of this process was investigated in vitro. The first step in transcription is binding of RNA polymerase to a promoter. Protein-DNA interaction can be studied by footprinting experiments. However, it is worth noting that interactions of DnaA molecules with weak DnaA boxes (especially at relatively low DnaA concentrations which are similar to those causing stimulation of pR) are unstable. They could not be detected by footprinting (15), but could by gel retardation (Fig. 6). Nevertheless, if DnaA stimulates binding of RNA polymerase to the pR promoter region, one might expect a more efficient protection of a promoter DNA fragment by RNA polymerase in the presence of DnaA than in the absence of this protein.
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When DNase I footprinting experiments were performed using relatively high RNA polymerase concentrations, efficient protection of the pR promoter region was observed, and addition of DnaA protein did not enhance this signal (Fig. 7 and data not shown). However, when the concentration of RNA polymerase was decreased to such a value that little protection of the pR region was observed under standard reaction conditions, addition of low amounts of DnaA resulted in an appearance of the pR region protection similar to that observed at high RNA polymerase concentrations (Fig. 7). Higher DnaA concentrations inhibited RNA polymerase binding, which is compatible with the results measuring the transcript levels from the pR promoter. We conclude that DnaA recruits RNA polymerase to the pR promoter, especially when both proteins are present at relatively low concentrations, which may resemble in vivo conditions.
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Promoter Escape, but Not Isomerization, Is Stimulated by DnaAUsing in vitro KMnO4 footprinting techniques, we found no significant changes in the kinetics of isomerization (formation of the open complex from the closed complex) at pR in response to the presence of various DnaA concentrations (data not shown). Therefore, we propose that the isomerization step is not affected by DnaA during stimulation of transcription from the pR promoter.
In contrast to isomerization, we found that the process of promoter clearance (promoter escape) is more efficient in the presence of low DnaA concentrations than in the absence of DnaA (Fig. 8). The effect was not dramatic but significant. Therefore, we conclude that DnaA activates transcription from the pR promoter by a double mechanism, i.e. by stimulation of RNA polymerase binding to the promoter region and by facilitating promoter clearance (promoter escape).
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Excess of DnaA Inhibits RNA Polymerase Binding to the pR Promoter RegionAs demonstrated previously, DnaA protein stimulates the pR promoter at low concentrations, whereas activity of this promoter is impaired at higher DnaA concentrations (8) (Figs. 3,4,5). We found that at moderately high concentrations of DnaA, interactions of RNA polymerase with the pR promoter region were significantly less efficient than in the absence of DnaA (Fig. 9). No protection by DnaA alone was observed at the weak DnaA boxes (Fig. 9). Therefore, we conclude that DnaA at high concentrations inhibits binding of RNA polymerase to pR. This inhibition is sufficient to account for DnaA-mediated repression of transcription from this promoter.
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DISCUSSION |
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Significantly more problematic, and more difficult to predict, was the mechanism of stimulation of transcription by DnaA. Here we demonstrate that the mechanism of DnaA-mediated activation of bacteriophage pR promoter is complicated and unusual. First, DnaA binds downstream of the activated promoter, which is uncommon in bacteria. Second, DnaA stimulates both binding of RNA polymerase to the promoter region and promoter clearance. This is also peculiar as most transcription activators either recruit RNA polymerase to a particular promoter or stimulate the isomerization process (i.e. formation of the open complex) (25, 26). Heparin could potentially interact with DnaA and then affect the interpretation of the results of promoter clearance experiments. But even in such a case, we would underestimate effects of DnaA rather than overestimate them. In addition, we did experiments estimating promoter clearance efficiency without heparin, and results analogous to those depicted in Fig. 8 were obtained (data not shown). Furthermore, when in vitro run-off transcription experiments were performed in the presence and absence of heparin, stimulation of the pR promoter activity was observed exactly at the same DnaA concentrations in both types of experiments (Figs. 3 and 4, and data not shown). These results strongly suggest that interaction of heparin with DnaA is weak, if any, and it is not significant for DnaA-mediated transcription activation.
Two weak DnaA boxes located downstream of the pR promoter are necessary and sufficient for transcription stimulation. It seems that weak interaction of DnaA with these regions is important, as replacement of the most proximal weak DnaA box with the consensus DnaA box sequence (ideal DnaA box) resulted in significant impairment of the promoter activation. Interestingly, plasmids bearing the ideal DnaA box 1 could transform E. coli cells efficiently. Clearly, the transformation assay is a less sensitive test to assess the transcription activator function of DnaA than in vitro transcription experiments. Moreover, a competition between DnaA boxes located on E. coli chromosome and plasmid DNA for DnaA protein binding may be significantly more complicated in vivo than in our in vitro assays, where only several DnaA-binding sequences were present on one DNA molecule. Therefore, precise prediction of amounts of DnaA protein available for binding to box 1 and box 2 in bacterial cells is impossible at this stage of research. Finally, one cannot exclude an influence of DNA topology on the efficiency of DnaA-mediated stimulation of pR activity, especially at various concentrations of this protein. In this light, it is worth noting that in our in vitro assays we used linear DNA templates whereas plasmids employed in the transformation assay occur in cells as circular, superhelically twisted molecules. Although efficiency of DnaA-mediated stimulation of pR observed in vivo during gene fusion analysis is generally similar to that measured in in vitro transcription assays at optimal DnaA concentrations (8), this parameter might vary in details between linear and circular DNA templates.
The requirement for two weak DnaA boxes to activate pR may be explained by the finding that for binding of DnaA protein to each particular weak DnaA box the presence of at least two DnaA-binding sequences is absolutely necessary (1, 2, 27). These boxes may be separated even by a few hundred bp (27), and in fact, the boxes 1 and 2 (Fig. 1) are separated by about 200 bp. Interestingly, increase in the distance between the transcription start site and the proximal DnaA box from 18 to 23 bp (i.e. by a half of the helical turn) did not affect DnaA-mediated regulation of pR. Further increase in this distance made the activation less effective, which indicates that the distance from the promoter to the proximal DnaA box, rather than a precise location of the bound DnaA protein at either side of the DNA helix relative to a promoter-bound RNA polymerase, is crucial. On the other hand, inversion of one of the two DnaA boxes had some effects on the transcription stimulation, suggesting that proper arrangement of DnaA protein molecules may, nevertheless, play a role in the activation of pR.
Our previous electron microscopic studies revealed that DnaA boxes 1 and 2 are not the preferential DnaA-binding sites on the DNA fragment encompassing the replication region, although some binding to those regions was unambiguously documented (14). In addition, using a DNA template of different length, an unambiguous DnaA binding to box 1 and box 2 was observed in electron microscopic experiments, provided that both boxes were present (27). This corroborates the demonstration here that these DnaA boxes are necessary and sufficient for DnaA-mediated stimulation of the pR promoter activity.
In conclusion, results presented in this report led us to propose the molecular mechanism of DnaA-mediated stimulation of pR activity. We suggest that the activation of the pR promoter by DnaA occurs at two steps. First, binding of DnaA to the two weak DnaA boxes stimulates binding of RNA polymerase. In the second step, RNA polymerase clearance is enhanced. The two boxes are separated by about 200 bp, and binding is likely to be cooperative, as suggested previously (27), and by the rules derived from the binding of DnaA protein (1, 2, 28). This may include formation of a higher order nucleoprotein structure, mediated by DNA looping. Formation of such a structure could be necessary for proper arrangement of DnaA molecules and their interactions with RNA polymerase. Such interactions (particularly with the subunit) were suggested previously (8) and might be responsible for recruitment of RNA polymerase to the pR promoter region. Moreover, formation of the higher order nucleoprotein structure might result in a partial denaturation of the DNA helix near pR, which could stimulate promoter clearance.
We suggest that it is important during development that activation of pR occurs by low DnaA concentrations at low affinity DnaA boxes. In this way, early
DNA replication, which proceeds according to the
mode, is stimulated by DnaA, and when DnaA becomes limiting the switch to
-mode of replication may occur (12).
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FOOTNOTES |
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¶ Supported by Alexander von Humboldt Foundation Fellowship IVPOL 1063505 STP.
|| Supported by the Fonds der chemischen Industrie (Germany).
Supported by Foundation for Polish Science subsidy 14/2000. To whom correspondence should be addressed: Dept. of Molecular Biology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland. Tel.: 48-58-346-3014; Fax: 48-58-301-0072; E-mail: wegrzyn{at}biotech.univ.gda.pl.
1 The abbreviation used is: DTT, dithiothreitol.
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REFERENCES |
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