| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany
Correspondence
Rolf Wagner
R.Wagner{at}rz.uni-duesseldorf.de
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Present address: Mediwiss Analytic GmbH, Moers, Germany.
A supplementary figure showing the sequences of the promoter constructs used in this study is available with the online version of this paper.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Mitchell et al., 2003; Shultzaberger et al., 2007; Vicente et al., 1991), the DNA geometry in the vicinity of the core promoter is generally of prime importance (Geiselmann, 1997). A number of bacterial proteins, termed nucleoid-associated proteins (NAPs) interact with DNA in a non-sequence-specific manner. NAPs bind preferentially to curved or flexible DNA, causing DNA compaction and structural rearrangements during DNA transactions (Dame, 2005; Luijsterburg et al., 2006). Some NAPs may recognize degenerated consensus sequences (Bouffartigues et al., 2007; Shao et al., 2008; Cui et al., 1995). For many genes, these proteins can affect transcriptional efficiency directly through alteration of the DNA architecture in the vicinity of promoters (Rochman et al., 2004). Often, DNA curvature and binding of regulatory proteins are synergistic elements in transcription regulation, and are strongly affected by the angular orientation with respect to the promoter core elements (Wu & Crothers, 1984). Hence, the DNA conformation and the exact geometry of bound transcription factors are crucial elements for the efficiency of many bacterial promoters (Jáuregui et al., 2003; Olivares-Zavaleta et al., 2006; Plaskon & Wartell, 1987).
In order to better understand the influence of DNA structure and protein-mediated regulation of promoters we have replaced the natural upstream sequences of the Escherichia coli rRNA promoters rrnB P1 and P2 by a synthetic curved DNA segment, containing sets of A:T tracts in helical phase, which results in a static curvature. Such curved DNA structures are known to be important for the binding of NAPs, such as H-NS and LRP or FIS (Yamada et al., 1990). Two sets of promoter variants were constructed, in which the distance of the curved DNA element was varied by about two helical turns. Our study shows that the basal transcriptional activity in the absence of proteins varies for both promoters as a function of the angular orientation of the curved DNA segment. As expected, all three proteins bind specifically to the curved DNA module. We also found, as has been shown previously, that LRP additionally binds to the P1 core promoter region, although with minor affinity.
To investigate the contribution of the distance and angular orientation to transcription factor-dependent regulation we performed in vitro transcriptional analysis in the presence of H-NS and LRP. We found that H-NS and LRP act as repressors for both promoters. For all constructs, H-NS-dependent repression did not vary substantially as a function of the distance and angular orientation of the synthetic binding site. In contrast, binding of LRP to the promoter variants caused repression in a helical-phase-dependent manner. Repression was maximal when LRP had been bound on the same face of the helix axis as the starting nucleotide. This effect was strong for the P2 promoter, and less pronounced for P1.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
The 92 bp synthetic curved DNA sequence CGA(GCCCGTTTTT)8GCCCTCTTG was obtained by HinfI restriction of plasmid pMG4A, kindly provided by M. Gastens, Heinrich-Heine-Universität Düsseldorf, and blunt ends were generated by mung bean nuclease treatment. It was then ligated at different distances from the P2 promoter. For this, pUC18-P2 was linearized with the restriction enzymes SphI, PstI, SalI, XbaI or HincII, followed by mung bean nuclease treatment except for the HincII-cleaved DNA. The SalI-digested DNA was separately filled-in with Klenow polymerase (Promega). The different plasmids pUC18-P2-1, pUC18-P2-2, pUC18-P2-3, pUC18-P2-4, pUC18-P2-5 and pUC18-P2-6 were then obtained by blunt-end ligation of the 92 bp bending module to the different vector constructs, so that the sequence given above represents the non-template strand. Construction of the pUC18-P1 derivatives was accomplished similarly, starting with pUC18-P1, yielding plasmids pUC18-P1-1 to pUC18-P1-5. The control promoter fragments without upstream curvature P1-K (176 bp) and P2-K (178 bp) were obtained by PvuII/SmaI restriction of pUC18-P1 and pUC18-P2, respectively. The sequences of all constructs were verified by sequencing and are shown in Supplementary Fig. S1.
Analysis of protein–DNA complexes by gel retardation.
DNA fragments were obtained by PvuII/XmaI digestion of the pUC18-P1 and pUC18-P2 derivatives. These fragments were purified by agarose gel electrophoresis and end-labelled by Klenow polymerase incorporation of [
-32P]dCTP. DNA-binding proteins were isolated and purified as described previously (Hillebrand et al., 2005; Pul et al., 2005). Protein–DNA complexes were formed in a buffer containing 50 mM Tris/HCl, pH 7.4, 70 mM KCl, 15 mM NaCl, 1 mM EDTA, 10 mM β-mercaptoethanol and 50 ng heparin µl–1. Standard reaction mixtures contained 2 nM DNA and the binding proteins LRP and/or H-NS at the concentrations indicated for the individual experiments.
In vitro transcription experiments.
E. coli RNA polymerase was isolated following published procedures (Burgess & Jendrisak, 1975; Gonzalez et al., 1977). Transcription experiments were performed as pseudo-single-round reactions as described elsewhere (Hsu, 1996). NAPs were added at the indicated concentration together with the RNA polymerase. Briefly, initiation complexes were formed in a total volume of 50 µl with 9 nM active RNA polymerase saturated with 
70 in the presence of 3 nM template DNA and the initiating nucleotides ATP (500 µM) and CTP (50 µM) in a buffer containing 50 mM Tris/acetate, pH 8.0, 10 mM magnesium acetate, 0.5 mM DTT, 0.1 mM EDTA, 100 µg acetylated BSA ml–1 and 80 mM potassium glutamate. [-32P]UTP [20 µCi (740 kBq)] [3000 Ci mmol–1 (111 TBq mmol–1)] was added to the mixture, followed by 20 min incubation at 30 °C. Initiation was stopped by the addition of 50 ng heparin µl–1, and after 5 min incubation at 30 °C all four NTPs were added (500 µM each), followed by a 5 min elongation reaction. A chase mixture (2 mM each NTP, 2 mg heparin ml–1) was added and samples were incubated for another 5 min at 30 °C. The samples were put on ice and the reaction was stopped by adding formamide sample buffer, which contained a defined amount of a radiolabelled loading standard. Transcription products were separated on 10 % denaturing polyacrylamide gels and visualized by autoradiography.
DNase I footprinting.
Limited hydrolysis of free DNA and protein–DNA complexes was performed with the corresponding P1 or P2 promoter fragments and different H-NS or LRP concentrations. DNA fragments were end-labelled by Klenow polymerase incorporation of [-32P]dCTP. Samples with or without protein were incubated in the presence of 0.5 mU DNase I µl–1 for 30 s at 25 °C. Hydrolysis was stopped by addition of 330 mM sodium acetate, pH 4.8, 10 mM EDTA and 10 ng glycogen µl–1, followed by phenol extraction. For the sequence assignment, G- and A-specific chemical cleavage reactions were performed as described elsewhere (Maxam & Gilbert, 1980). Cleavage products were separated on denaturing 8 % polyacrylamide gels and visualized by autoradiography.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Yamada et al., 1990). Therefore, we constructed a synthetic NAP-binding domain consisting of eight repetitive AT-clusters that were separated by GC sequences, placing the AT-tracts in helical register. The curved nature of the bending module was verified by gel electrophoresis at different temperatures and confirmed by in silico analysis, employing the DNA structure prediction program DIAMOD (Dlakic & Harrington, 1998). The DNA fragment exhibited notable curvature with a k-value of 1.73 and an approximate bending angle of 10 ° (Thompson & Landy, 1988). The 92 bp bending module was ligated upstream of the rrnB P1 and P2 promoters at different distances relative to the respective transcription start sites. The distances of the bending module centre and the transcription start sites varied between 87 and 107 bp, which corresponds to a phasing of the curved DNA module of almost two helical turns (Fig. 1). The resulting constructs were designated P1-1 to P1-5 and P2-1 to P2-6, respectively, depending on the particular promoter (P1 or P2), to which the modules were fused (Fig. 1).
|
). Hence, effects on promoter binding and transcriptional activity due to interaction of a potential UP element with the -CTD of RNA polymerase can be excluded from this study.
Predicted DNA conformation of the different rrnB P1 and P2 promoter variants
As a reliable algorithm for the curvature prediction of the constructs employed in this study, we chose the program DIAMOD, which allows the application of different model calculations for curvature prediction and includes a versatile method for visualization of the calculated structures (Dlakic & Harrington, 1998).
Fig. 2(a, b) shows the predicted structures of the different phasing constructs, which were calculated according to the Gabrielian trinucleotide parameters (Gabrielian & Pongor, 1996). The spatial orientations of the different fragments are presented such that the promoter core regions and downstream sequences are arranged in the same plane and viewed from the same direction. It can immediately be recognized from the structure predictions that the different distances between the centres of the bending module (marked by the left-pointing arrows) to the transcription start site (marked by the kinked arrows) lead to different angular orientations. From the visual inspection it appears that at both promoters the constructs P1-1 and P1-3 (Fig. 2a) and P2-1 and P2-3 (Fig. 2b) have nearly the same orientation of the two centres (shown by the arrows). This aspect of the helix orientation is also consistent with the arrangement depicted in Fig. 1
, where B-DNA parameters have been used to indicate the angular orientation between the centre of the bending modules and the transcription start sites of the different constructs.
|
; Zacharias et al., 1992), no comparable observations have been made for the P2 promoter, which in its natural context completely lacks curved upstream sequences, although a weak UP element has been reported for rrnB P2 (Murray et al., 2003). Fig. 2(c, d) shows the results of in vitro transcription analysis with the different P1 and P2 constructs. The orientations of the curved upstream regions had a strong effect on the basal transcriptional activity of the two promoters. Activity was highest when the inside of the curved DNA structure was in the same plane as the transcription start site (Fig. 2). The angular orientation of the curved sequence appeared to be even more important than its distance from the core promoter. This can be inferred by comparison of the transcriptional activity of P1-1 and P1-3 (and also P2-1 and P2-3), which have different distances of the bending centre relative to the promoter, although the orientations are similar (see also Fig. 1).
In summary, it can be concluded that the influence of curved upstream sequences on the intrinsic promoter activity is phase-of-the-helix-dependent. One explanation for the different promoter activities may be the preferred orientation of upstream sequences, such that they wrap around RNA polymerase in a left-handed way, so that RNA polymerase is positioned at the inside of the curved DNA. This conclusion confirms the notion of the phase-of-the-helix dependence of upstream sequences at the rrnB P1 promoter (Meng et al., 2001) and demonstrates that the observation is also valid for the P2 promoter constructs.
Binding of H-NS and LRP to promoter fragments without upstream curvature
At first, we had to verify that the curved DNA module functioned as an efficient binding site for the transcription factors H-NS and LRP, and determine whether or not the P1 and P2 promoter DNA fragments without the upstream bending module (P1-K and P2-K, respectively) were able to interact with the different proteins. The P1-activating protein FIS was used additionally as a control. Gel retardation analysis with the corresponding P1 and P2 promoter fragments, containing only the promoter elements without the curved DNA region, revealed, consistent with previous analyses, that LRP binds within the P1 core promoter element (Kd >2 µM), while no complex formation occurred with P2-K (Fig. 3). Moreover, H-NS did not bind specifically to either of the two fragments without the bending module, supporting the recent finding that the curved DNA region of the P1 promoter acts as a nucleation site for H-NS binding. Only at sufficiently high concentrations (>3 µM), after initial binding to a curved DNA target site, was H-NS able to polymerize into the core promoter region. The retardation analysis of promoter fragments without the bending module thus confirmed our previous results, namely that efficient H-NS binding requires a curved DNA region of the rrn P1 promoter (Pul et al., 2005, 2007). Moreover, we found that the transcription factor FIS binds to the genuine rrnB P2 core promoter region (data not shown), which implies repression and therefore indicates the potential for differential regulation of the two rRNA promoters (Liebig & Wagner, 1995).
|
). Hence, the fusion of a synthetic curved AT-rich sequence leads to specific binding of H-NS and LRP to promoter-containing target DNA fragments. The known binding cooperativity of H-NS (Pul et al., 2005) is supported by the fact that no intermediate complex bands were observed in the binding studies. It can be seen that the mobilities of the H-NS–DNA complexes varied slightly for the different constructs, which reflects the different positions of the bending module with respect to the fragment ends. Moreover, the stability of the different complexes was not completely uniform, as can be inferred from some of the diffuse bands in Fig. 4.
|
4 µM) and LRP (Kd <0.5 µM) for each individual fragment did not vary noticably, however, which indicates that the affinity for the initial binding sites of a given fragment is independent of the relative position of the binding module. Analysis of the binding of LRP to the different P1 constructs revealed, however, that the number of individual complexes, their cooperativity and the mobility of the different LRP–DNA complexes varied for the different fragments. Hence, for the P1 constructs the position of the curvature with respect to the core promoter affects the cooperativity of LRP binding. This can be seen when complexes from the constructs P1-1 and P1-3 are compared with the other DNA fragments (Fig. 4). For these constructs, the formation of a second complex with lower mobility, indicative of looping or wrapping, could be observed at low LRP concentrations. Interestingly, constructs P1-1 and P1-3 had very similar helical phasing geometry, while the distance between the bending module and the transcription start site for these two fragments differed by 10 bp, corresponding almost exactly to one full helical turn (Fig. 1a). Formation of such a higher-order complex is obviously enhanced by the spatial orientation of the core promoter and the synthetic bending module, which appears to be optimal for the constructs P1-1 and P1-3. The centre of curvature and the core promoter region are on the same face of the DNA helix, facilitating cooperative protein interactions. Such a steric arrangement is also supported by the structure predicted in Fig. 2(a). The LRP-binding studies with the P2 constructs, which do not have a second protein-binding site within the core promoter structure, did not reveal evidence for higher-order complexes, supporting the conclusion that cooperativity likely results from protein contacts at a distance when placed at the same face of the DNA helix. Our results support the view that the helical phase matters more than the lateral distance, because neither a shorter nor a larger distance revealed comparable binding properties. As will be seen below, the same is true for the transcriptional activity of the different constructs.
Interestingly, the simultaneous presence of H-NS and LRP results in supershifted complexes on retardation gels (Fig. 4a, b; lanes 7), indicating that binding of both proteins is not mutually exclusive but rather occurs synergistically. Such synergism between H-NS and LRP has been observed in previous studies (Pul et al., 2005).
In summary, our results are consistent with the notion that efficient H-NS binding requires a natural AT-rich curved sequence element, such as that present upstream of all rRNA P1 promoters (upstream activation sequence; UAS) (Lindahl & Zengel, 1986). Note that this curved AT-rich UAS region should not be mistaken by the so-called UP element, which is known to interact directly with the -CTD of RNA polymerase. The natural UAS region can effectively be replaced by a synthetic bending module, as shown in this study. Likewise, any high-affinity binding of LRP requires a potential UAS region or a bending module, even if the P1 core promoter apparently contains a low-affinity LRP-binding site (Kd >2 µM), which only exhibits LRP binding at high concentrations. The fusion of the synthetic bending module converts the two DNA fragments with the rrnB promoters P1 and P2 to primary LRP target sites with Kd values of about 0.5 µM.
DNase I footprint analysis
To verify the locations and intensities of H-NS and LRP binding to the fusion constructs, we performed DNase I footprinting analysis at various protein concentrations. A representative example for the non-template strands of the constructs P1-2 and P2-2 is shown in Fig. 5. Note that different protein concentrations were applied for LRP and H-NS, in accordance with the different binding affinities of the two proteins. Both fragments showed clear protection throughout the sequence of the bending module. Although this sequence is identical in both constructs, a significantly stronger protection in the presence of H-NS could be detected for the P1 fragment. This is in line with the stronger H-NS-dependent repression of transcription observed for the P1 compared with the P2 fusion constructs shown below (Figs 7b and 8b). Interestingly, significant binding of H-NS to the P1 promoter core region was also apparent from the corresponding DNase I protection pattern, suggesting that, in case of the P1 promoter fragment, increased inhibition results from the cooperative interaction of two distant H-NS-binding sites. Note that no pattern of comparable H-NS-dependent protection was apparent for the promoter core region of the P2 fragment. A single hyperreactive site, which is rarely detected in H-NS footprints, might indicate an H-NS-dependent widening of the small groove on the outside of the curved bending module, giving rise to better accessibility for the nuclease.
|
|
|
).
Effects of H-NS and LRP on the transcriptional activity of the different promoter fragments without upstream curvature
The effects of LRP and H-NS on the two promoter DNA fragments P1 K and P2 K without curved upstream regions were also analysed under in vitro transcription conditions (Fig. 6). These experiments showed that the P1 core promoter was strongly inhibited in the presence of LRP (nearly 90 % inhibition at 2 µM LRP), but only weak inhibition was observed for P2 (Fig. 6a). The results support the findings of previous binding and DNase I footprint analyses, which demonstrate that LRP can bind at the rrnB P1 promoter between the –35 and –10 regions (Fig. 3; Pul et al., 2007). In the P2 construct, which lacks a binding site upstream of the promoter core region, inhibition at 2 µM LRP was reduced to 40 % (Fig. 6b). Transcription in the presence of H-NS was inhibited for both promoters to an identical extent (40 % inhibition at 2 µM H-NS). Apparently, there is transient interaction of H-NS with the core promoter DNA, but this interaction is not stable enough to survive gel electrophoresis during the binding assays.
|
In vitro transcription reactions were performed in the presence of different concentrations of the two transcription factors. Results for the P1 promoter constructs are summarized in Fig. 7. Representative gels for the analysis in the presence of H-NS and LRP are presented in Fig. 7(a, c), respectively. In Fig. 7(b, and d) the quantification of three independent experiments is summarized as bar diagrams. It can be seen that the presence of H-NS caused considerable inhibition of the basal transcription (40–60 % at 1 µM and about 90 % at 2 µM). The degree of inhibition did not deviate significantly, however, for the different phasing constructs (Fig. 7b). This observation is consistent with the mechanism often proposed for H-NS-dependent transcriptional inhibition, which is based on oligomerization of the protein along the DNA, thereby preventing RNA polymerase from binding to the promoter.
The effect of LRP on the P1 constructs is shown in Fig. 7 (c, d). The inhibition in the presence of LRP was stronger than that observed for H-NS (60–90 % at 1 µM and > 90 % at 2 µM), which is in line with the stronger binding affinity of LRP and with the existence of a low-affinity LRP-binding site within the core promoter region. A slight effect of the phasing of the binding site was apparent at the lower LRP concentration. Construct P1-3, which has its centre of curvature opposite the transcription start site, showed maximal inhibition. This might indicate a low degree of synergism between the two LRP-binding sites.
The same analysis was performed with the P2 phasing constructs. Results are shown in Fig. 8. Compared with the curvature-free P2 promoter fragment (P2 K), inhibition by both proteins was stronger for fragments that contained the curvature (compare Fig. 8 with Fig. 6 at 1 and 2 µM protein). Quantitative evaluation of H-NS-dependent inhibition revealed that it was independent of the location of the binding sites. In the presence of 1 µM H-NS the relative inhibition reached an average of 30 %, and 70 % in the presence of 2 µM H-NS (Fig. 8a, b). These results support the view that H-NS-mediated inhibition occurs by occlusion of the promoter region through oligomerization of H-NS proteins along the promoter region, irrespective of angular effects.
While inhibition in the presence of LRP was likewise enhanced compared with the curvature-free P2 DNA fragment, the extent of inhibition clearly depended on the position and angular orientation of the protein-binding site (Fig. 8d). The degree of inhibition for the different P2 constructs varied periodically with increasing distance between the protein-binding site and the core promoter. It was maximal for constructs P2-2 and P2-5, and approached a minimum for constructs P2-1, P2-3 and P2-6. Comparison of the angular orientation of the centre of the binding site with respect to the transcription start site for the different constructs revealed that P2-2 and P2-5 were located at the same face of the DNA helix as the start position, while P2-1 and P2-6 were further apart, and P2-3 had almost the opposite orientation (Fig. 1b). This oscillation in promoter activity for the different P2 promoter constructs was visible at both transcription factor concentrations (1 and 2 µM), although it was more pronounced at 1 µM. The results clearly underline the importance of the angular orientation of regulatory LRP-binding sites to the relative promoter efficiency.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Hirvonen et al., 2001; Pul et al., 2005, 2007). In E. coli all rRNA P1 promoters are preceded by a curved regulatory DNA region responsible for transcription factor binding. The binding sites of these proteins are located in the AT-rich sequences found upstream of all rrn P1 promoters. These regions are known as UASs, because they enhance P1 activities both in vivo and in vitro (Gosink et al., 1993; Leirmo & Gourse, 1991; Ross et al., 1990; Zacharias et al., 1992). The seven E. coli rRNA operon UAS regions differ in structure and transcription factor-binding efficiencies (Hillebrand et al., 2005; Pul et al., 2005), and contribute to the differential regulation of rRNA transcription, in both the presence and the absence of transcription factors. The mechanisms of regulation mediated by the NAPs are not unique but include alterations of DNA structure and topology, protein–protein interaction and competition with RNA polymerase binding (Afflerbach et al., 1999; Aiyar et al., 2002; Bokal et al., 1997; Dame et al., 2002; Muskhelishvili et al., 1995; Pul et al., 2005; Schröder & Wagner, 2000). Although topological effects cannot be excluded (Travers & Muskhelishvili, 1998), FIS activates the rrnB P1 promoters by binding upstream of the promoter and interacting with the subunit of RNA polymerase (Bokal et al., 1997). In contrast, LRP binding inhibits transcription from P1 promoters via the formation of higher-order looped nucleoprotein complexes, including the core promoter region. H-NS binds to a nucleation site located in the UAS region and polymerizes along the DNA, reaching into the promoter region. In the case of rrnB P1, a trapping mechanism has been demonstrated that locks RNA polymerase in the apex of a DNA loop formed by promoter-flanking regions bridged by H-NS oligomers (Dame et al., 2002; Schröder & Wagner, 2000).
We have shown here that the intrinsic basal activity of the natural P1 and P2 promoters of the rrnB operon depends on the presence of an upstream bending module. Variations of the distance of this curved DNA from the core promoter result in different orientations of the centre of the bending module with respect to the transcription start site. Comparison of the activities of the constructs P1-1 and P1-3 shows that the angular orientation of the bending module is obviously of a similar importance as the distance to the promoter region. This seems to be valid even for a promoter that, in its natural context, is not preceded by a curved upstream region, as shown here for P2. The increase in promoter activity likely results from extended RNA polymerase–DNA contacts due to DNA wrapping and concomitant facilitated open complex formation (Cellai et al., 2007; Davis et al., 2005).
We have deliberately constructed the promoter variants without UP element sequences in order not to complicate the analysis by additional stabilization of RNA polymerase initiation complexes via the C-terminal domain of the -subunits (-CTD). While such an interaction is of known significance for the activity of rRNA P1 promoters, the P2 promoters in their natural context do not share such an interaction. Because we were only interested in the effects of curvature and position relative to the core promoter, and because neither H-NS nor LRP is known to bind to UP element sequences, none of the linker sequences between the core promoters and the bending module of the constructs employed in this study contains a sequence similar to the published UP element consensus: –59nnAAA(AyT)(AyT)T(AyT)TTTTnnAAAAnnn–38 (Estrem et al., 1998).
Basal transcription activities were repressed in the presence of H-NS for both promoter constructs. This argues for the existence of a silencing mechanism whereby H-NS initially binds to the upstream bending modules and polymerizes towards the core promoter region. H-NS-dependent repression is more pronounced for P1 constructs due to additional weak interaction of H-NS with the P1 promoter core region, as shown by the in vitro transcription and DNase I experiments. Binding of H-NS to the promoter core alone in the absence of the bending module does not cause significant inhibition, however. In contrast, LRP shows a distinct mode of repression. Our study shows that LRP binds directly to the P1 core promoter in the absence of the curved DNA module. No comparable interaction can be observed with the P2 core promoter. This argues for a differential regulation of the two rrn promoters. In contrast to H-NS, LRP-dependent inhibition shows a clear phasing characteristic for the P2 constructs with maximal inhibition at an angular orientation of the binding centre on the same face of the helix as the transcription start site. This phasing is less clear for the P1 constructs. We assume that a mechanism exists whereby the orientation of the LRP–nucleoprotein structure differentially interferes with RNAP binding to the promoter region. The phasing dependence of the LRP repression is impaired for the P1 constructs due to additional, though weak, direct binding of LRP to the core promoter region. Overall, this leads to a stronger inhibition of the P1 constructs in the in vitro transcription assays.
In summary, both H-NS and LRP require curved or flexible upstream DNA elements in order to bind with high affinity to the rrnB promoters. Their effects on transcription are not identical, however. The two proteins have different modes of action: whereas H-NS leads to silencing of promoter activity by coating the DNA, LRP causes looped or wrapped DNA structure, attenuating RNA polymerase binding to the promoter. The latter depends on the orientation of the upstream sequence relative to the core promoter region.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: J. W. B. Moir
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Aiyar, S. E., McLeod, S. M., Ross, W., Hirvonen, C. A., Thomas, M. S., Johnson, R. C. & Gourse, R. L. (2002). Architecture of FIS-activated transcription complexes at the Escherichia coli rrnB P1 promoters. J Mol Biol 316, 501–516.[CrossRef][Medline]
Beloin, C., Jeusset, J., Revet, B., Mirambeau, G., Le Hegarat, F. & Le Cam, E. (2003). Contribution of DNA conformation and topology in right-handed DNA wrapping by the Bacillus subtilis LrpC protein. J Biol Chem 278, 5333–5342.
Bokal, A. J., Ross, W., Gaal, T., Johnson, R. C. & Gourse, R. L. (1997). Molecular anatomy of a transcription activation patch: FIS–RNA polymerase interactions at the Escherichia coli rrnB P1 promoter. EMBO J 16, 154–162.[CrossRef][Medline]
Bouffartigues, E., Buckle, M., Badaut, C., Tracers, A. & Rimsky, S. (2007). H-NS cooperative binding to high-affinity sites in a regulatory element results in transcriptional silencing. Nat Struct Mol Biol 14, 441–448.[CrossRef][Medline]
Burgess, R. R. & Jendrisak, J. J. (1975). A procedure for the rapid, large scale purification of E. coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA cellulose chromatography. Biochemistry 14, 4634–4638.[CrossRef][Medline]
Cellai, S., Mangiarotti, L., Vannini, N., Naryshkin, N., Kortkhonjia, E., Ebright, R. H. & Rivetti, C. (2007). Upstream promoter sequences and CTD mediate stable DNA wrapping within the RNA polymerase–promoter open complex. EMBO Rep 8, 271–278.[CrossRef][Medline]
Cui, Y., Wang, Q., Stormo, G. D. & Calvo, J. M. (1995). A consensus sequence for binding of LRP to DNA. J Bacteriol 177, 4872–4880.
Dame, R. T. (2005). The role of nucleoid-associated proteins in the organization and compaction of bacterial chromatin. Mol Microbiol 56, 858–870.[CrossRef][Medline]
Dame, R. T., Wyman, C., Wurm, R., Wagner, R. & Goosen, N. (2002). Structural basis for H-NS-mediated trapping of RNA polymerase in the open initiation complex at rrnB P1. J Biol Chem 277, 2146–2150.
Davis, C. A., Capp, M. W., Record, M. T., Jr & Saecker, R. M. (2005). The effects of upstream DNA on open complex formation by Escherichia coli RNA polymerase. Proc Natl Acad Sci U S A 102, 285–290.
Dlakic, M. & Harrington, R. E. (1998). DIAMOD: display and modeling of DNA bending. Bioinformatics 14, 326–331.
Estrem, S. T., Gaal, T., Ross, W. & Gourse, R. L. (1998). Identification of an UP element consensus sequence for bacterial promoters. Proc Natl Acad Sci U S A 95, 9761–9766.
Gabrielian, A. & Pongor, S. (1996). Correlation of intrinsic DNA curvature with DNA property periodicity. FEBS Lett 393, 65–68.[CrossRef][Medline]
Geiselmann, J. (1997). The role of DNA conformation in transcriptional initiation and activation in Escherichia coli. Biol Chem 378, 599–607.[Medline]
Gonzalez, N., Wiggs, J. & Chamberlin, M. J. (1977). A simple procedure for resolution of E. coli RNA polymerase holoenzyme from core polymerase. Arch Biochem Biophys 182, 404–408.[CrossRef][Medline]
Gosink, K. K., Ross, W., Leirmo, S., Osuna, R., Finkel, S. E., Johnson, R. C. & Gourse, R. L. (1993). DNA binding and bending are necessary but not sufficient for Fis-dependent activation of rrnB P1. J Bacteriol 175, 1580–1589.
Hillebrand, A., Wurm, R., Menzel, A. & Wagner, R. (2005). The seven E. coli ribosomal RNA operon upstream regulatory regions differ in structure and transcription factor binding efficiencies. Biol Chem 386, 523–534.[CrossRef][Medline]
Hirvonen, C. A., Ross, W., Wozniak, C. E., Marasco, E., Anthony, J. R., Aiyar, S. E., Newburn, V. H. & Gourse, R. L. (2001). Contributions of UP elements and the transcription factor FIS to expression from the seven rrn P1 promoters in Escherichia coli. J Bacteriol 183, 6305–6314.
Hsu, L. M. (1996). Quantitative parameters for promoter clearance. Methods Enzymol 273, 59–71.[Medline]
Jáuregui, R., Abreu-Goodger, C., Moreno-Hagelsieb, G., Collado-Vides, J. & Merino, E. (2003). Conservation of DNA curvature signals in regulatory regions of prokaryotic genes. Nucleic Acids Res 31, 6770–6777.
Leirmo, S. & Gourse, R. L. (1991). Factor-independent activation of Escherichia coli rRNA transcription. I. Kinetic analysis of the roles of the upstream activator region and supercoiling on transcription of the rrnB P1 promoter in vitro. J Mol Biol 220, 555–568.[CrossRef][Medline]
Liebig, B. & Wagner, R. (1995). Effects of different growth conditions on the in vivo activity of the tandem Escherichia coli ribosomal RNA promoters P1 and P2. Mol Gen Genet 249, 328–335.[CrossRef][Medline]
Lindahl, L. & Zengel, J. M. (1986). Ribosomal genes in Escherichia coli. Annu Rev Genet 20, 297–326.[CrossRef][Medline]
Luijsterburg, M. S., Noom, M. C., Wuite, G. J. & Dame, R. T. (2006). The architectural role of nucleoid-associated proteins in the organization of bacterial chromatin: a molecular perspective. J Struct Biol 156, 262–272.[CrossRef][Medline]
Maxam, A. M. & Gilbert, W. (1980). Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65, 499–560.[Medline]
Meng, W., Belyaeva, T., Savery, N. J., Busby, S. J. W., Ross, W., Gaal, T., Gourse, R. L. & Thomas, M. S. (2001). UP element-dependent transcription at the Escherichia coli rrnB P1 promoter: positional requirements and role of the RNA polymerase subunit linker. Nucleic Acids Res 29, 4166–4178.
Mitchell, J. E., Zheng, D., Busby, S. J. & Minchin, S. D. (2003). Identification and analysis of ‘extended –10’ promoters in Escherichia coli. Nucleic Acids Res 31, 4689–4695.
Murray, H. D., Appleman, J. A. & Gourse, R. L. (2003). Regulation of the Escherichia coli rrnB P2 promoter. J Bacteriol 185, 28–34.
Muskhelishvili, G., Travers, A. A., Heumann, H. & Kahmann, R. (1995). FIS and RNA polymerase holoenzyme form a specific nucleoprotein complex at a stable RNA promoter. EMBO J 14, 1446–1452.[Medline]
Olivares-Zavaleta, N., Jauregui, R. & Merino, E. (2006). Genome analysis of Escherichia coli promoter sequences evidences that DNA static curvature plays a more important role in gene transcription than has previously been anticipated. Genomics 87, 329–337.[CrossRef][Medline]
Plaskon, R. R. & Wartell, R. M. (1987). Sequence distributions associated with DNA-curvature are found upstream of strong E. coli promoters. Nucleic Acids Res 15, 785–796.
Pul, Ü., Wurm, R., Lux, B., Meltzer, M., Menzel, A. & Wagner, R. (2005). LRP and H-NS – cooperative partners for transcription regulation at E. coli rRNA promoters. Mol Microbiol 58, 864–876.[CrossRef][Medline]
Pul, Ü., Wurm, R. & Wagner, R. (2007). The role of LRP and H-NS in transcription regulation: involvement of synergism, allostery and macromolecular crowding. J Mol Biol 366, 900–915.[CrossRef][Medline]
Rochman, M., Blot, N., Dyachenko, M., Glaser, G., Travers, A. & Muskhelishvili, G. (2004). Buffering of stable RNA promoter activity against DNA relaxation requires a far upstream sequence. Mol Microbiol 53, 143–152.[CrossRef][Medline]
Ross, W., Thompson, J. F., Newlands, J. T. & Gourse, R. L. (1990). E. coli Fis protein activates ribosomal RNA transcription in vitro and in vivo. EMBO J 9, 3733–3742.[Medline]
Schröder, O. & Wagner, R. (2000). The bacterial DNA-binding protein H-NS represses ribosomal RNA transcription by trapping RNA polymerase in the initiation complex. J Mol Biol 298, 737–748.[CrossRef][Medline]
Shao, Y., Feldman-Cohen, L. S. & Osuna, R. (2008). Functional characterization of the Escherichia coli Fis–DNA binding sequence. J Mol Biol 376, 771–785.[Medline]
Shultzaberger, R. K., Chen, Z., Lewis, K. A. & Schneider, T. D. (2007). Anatomy of Escherichia coli 70 promoters. Nucleic Acids Res 35, 771–788.
Thompson, J. F. & Landy, A. (1988). Empirical estimation of protein-induced DNA bending angles: applications to 
site-specific recombination complexes. Nucleic Acids Res 16, 9687–9705.
Travers, A. & Muskhelishvili, G. (1998). DNA microloops and microdomains: a general mechanism for transcription activation by torsional transmission. J Mol Biol 279, 1027–1043.[CrossRef][Medline]
Vicente, M., Kushner, S. R., Garrido, T. & Aldea, M. (1991). The role of the ‘gearbox’ in the transcription of essential genes. Mol Microbiol 5, 2085–2091.[CrossRef][Medline]
Wu, H. M. & Crothers, D. M. (1984). The locus of sequence-directed and protein-induced DNA bending. Nature 308, 509–513.[CrossRef][Medline]
Yamada, H., Muramatsu, S. & Mizuno, T. (1990). An Escherichia coli protein that preferentially binds to sharply curved DNA. J Biochem 108, 420–425.
Zacharias, M., Göringer, H. U. & Wagner, R. (1992). Analysis of the Fis-dependent and Fis-independent transcription activation mechanism of the E. coli ribosomal RNA P1 promoter. Biochemistry 31, 2621–2628.[CrossRef][Medline]
Received 19 March 2008;
revised 12 May 2008;
accepted 16 May 2008.
This article has been cited by other articles:
|
|
 |
|
  M. A. De la Cruz, E. Merino, R. Oropeza, J. Tellez, and E. Calva The DNA static curvature has a role in the regulation of the ompS1 porin gene in Salmonella enterica serovar Typhi Microbiology, July 1, 2009; 155(7): 2127 - 2136. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
