Repressor induced site-specific binding of HU for transcriptional regulation
1997; Springer Nature; Volume: 16; Issue: 12 Linguagem: Inglês
10.1093/emboj/16.12.3666
ISSN1460-2075
Autores Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle15 June 1997free access Repressor induced site-specific binding of HU for transcriptional regulation Tsunehiro Aki Tsunehiro Aki Laboratory of Molecular Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Building 37, Room 2E16, 37 Convent Drive MSC 4255, Bethesda, MD, 20892-4255 USA Search for more papers by this author Sankar Adhya Corresponding Author Sankar Adhya Laboratory of Molecular Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Building 37, Room 2E16, 37 Convent Drive MSC 4255, Bethesda, MD, 20892-4255 USA Search for more papers by this author Tsunehiro Aki Tsunehiro Aki Laboratory of Molecular Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Building 37, Room 2E16, 37 Convent Drive MSC 4255, Bethesda, MD, 20892-4255 USA Search for more papers by this author Sankar Adhya Corresponding Author Sankar Adhya Laboratory of Molecular Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Building 37, Room 2E16, 37 Convent Drive MSC 4255, Bethesda, MD, 20892-4255 USA Search for more papers by this author Author Information Tsunehiro Aki1 and Sankar Adhya 1 1Laboratory of Molecular Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Building 37, Room 2E16, 37 Convent Drive MSC 4255, Bethesda, MD, 20892-4255 USA The EMBO Journal (1997)16:3666-3674https://doi.org/10.1093/emboj/16.12.3666 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Transcription from two overlapping gal promoters is repressed by Gal repressor binding to bipartite gal operators, OE and OI, which flank the promoters. Concurrent repression of the gal promoters also requires the bacterial histone-like protein HU which acts as a co-factor. Footprinting experiments using iron–EDTA-coupled HU show that HU binding to gal DNA is orientation specific and is specifically dependent upon binding of GalR to both OE and OI. We propose that HU, in concert with GalR, forms a specific nucleoprotein higher order complex containing a DNA loop. This way, HU deforms the promoter to make the latter inactive for transcription initiation while remaining sensitive to inducer. The example of gal repression provides a model for studying how a 'condensed' DNA becomes available for transcription. Introduction The catalysis and regulation of a variety of DNA transactions, such as site-specific recombination, transcription, replication and packaging, involves formation of higher order DNA–multiprotein complexes (Echols, 1986; Grosschedl et al., 1994). Proteins that bind to DNA with high specificity and play a principal role, as well as proteins that bind to DNA with broad specificity and play an architectural role, participate in forming such complexes (Nash, 1996). In Escherichia coli, the latter group includes HU, IHF, H-NS and Fis. Both the formation and the nature of the nucleoprotein complexes are determined by the ability of the architectural proteins to deform DNA and facilitate critical DNA–protein and protein–protein contacts, which are otherwise prohibitory (Adhya, 1989; Pérez-Martin et al., 1994; Nash, 1996). The structural determination of such complexes will help researchers understand how they function. In transcriptional regulation such structures must possess an additional feature: responsiveness to external regulatory signals. As described here, the multiple regulation of transcription initiation in the galactose (gal) operon in E.coli is such an apparatus (Adhya, 1996). The gal transcription starts from two overlapping promoters, P1 and P2, which are subject to, among others, negative control by Gal repressor (GalR). GalR represses both promoters in vivo by binding to two operators, OE and OI, which flank the two promoters (Musso et al., 1978; Adhya and Miller, 1979; Aiba et al., 1981; Irani et al., 1983; Kuhnke et al., 1986). It was proposed that repression requires an interaction between the two operator-bound repressors, and thereby formation of a loop of the intervening 113 bp DNA encompassing the promoters (Irani et al., 1983; Majumdar and Adhya, 1984). In contrast to the in vivo observation, GalR represses P1 inefficiently and activates P2 in vitro with purified components, and does not form the proposed DNA loop (Mandal et al., 1990; Choy and Adhya, 1992; Goodrich and McClure, 1992). Because of this discrepancy between the in vivo and in vitro observations, we predicted that efficient and coordinated repression of the two promoters requires another factor that acts by assisting GalR in looping the DNA (Choy and Adhya, 1992). We recently discovered and purified an additional factor that co-operates with GalR for the concomitant repression of two gal promoters in vitro and identified the accessory factor as the bacterial histone-like protein HU (Aki et al., 1996). The HU protein from E.coli is predominantly a heterodimer of α and β subunits, each with a molecular weight of 9 kDa. It is an abundant DNA-binding protein associated with the bacterial nucleoid (Rouviére-Yaniv, 1978; Rouviére-Yaniv and Kjeldgaard, 1979). HU is not known to show DNA sequence specificity and has a tendency to bind to distorted regions of DNA, e.g. kinks, bends, single-stranded gaps and cruciforms (Bianchi et al., 1989; Pontiggia et al., 1993; Bianchi, 1994; Bonnefoy et al., 1994; Castaing et al., 1995). HU also binds to a transcription terminator which has a dyad symmetry followed by a stretch of AT base pairs (see below). The terminator attracts HU very likely by forming a hairpin in supercoiled DNA. In most cases of DNA transactions involving HU, it acts architecturally by assisting spatially separated sequence-specific DNA-bound proteins to associate by binding to and bending the intervening DNA region (Craigie et al., 1985; Johnson et al., 1986; Bramhill and Kornberg, 1988; Hodges-Garcia et al., 1989; Ogawa et al., 1989; Wada et al., 1989; Baker and Mizuuchi, 1992; Hwang and Kornberg, 1992; Lavoie and Chaconas, 1990; Lavoie et al., 1991; Lavoie and Chaconas, 1994; Segall et al., 1994; Bètermier et al., 1995; Pérez-Martin and deLorenzo, 1995). We report here that in restoring repression of transcription to P1 and P2 by GalR, HU binds site-specifically between the two gal operators. HU binding is entirely dependent upon binding of GalR to the two operators and is sensitive to exogenous signal D-galactose, thus revealing a novel way by which a HU-containing higher-order complex responds to transcriptional regulation. Results gal repression requires HU In a purified transcription system, GalR activates the P2 promoter and represses, albeit less efficiently, the P1 promoter (Choy and Adhya, 1992; Goodrich and McClure, 1992; Choy et al., 1995a; Figure 1, lane 2). As demonstrated previously, the HU aids GalR to repress both gal promoters in vitro (Aki et al., 1996; Figure 1, lane 4). Since HU by itself does not alter the levels of gal transcripts, it acts as a co-factor in this system (Figure 1). The repression of gal transcription observed in the presence of HU is lifted by the addition of the inducer, D-galactose (lane 6). The coordinated repression of P1 and P2 requires the binding of GalR to both gal operators, OI and OE; mutation of either results in loss of the coordination (lanes 8 and 10). These results are entirely consistent with the in vivo observations. We have proposed that the repression is exerted by formation of an intricate nucleoprotein complex of higher-order structure that contains a DNA loop (Irani et al., 1983; Majumdar and Adhya, 1984; Choy et al., 1995b; Aki et al., 1996). Figure 1.Effect of GalR and HU on transcription initiation from gal promoters. In vitro transcription assays were conducted using supercoiled gal DNA template (2 nM) carrying wild type operators (lanes 1–6 and 11–14, OE+OI+) or mutant operators (lanes 7 and 8, OE+OI− and lanes 9 and 10, OE−OI+). The DNA template used in lanes 11–14 was P1−P2+. The concentrations of GalR were as indicated; HU was 80 nM; and D-galactose was 10 mM. RNA transcripts were analyzed by electrophoresis on an 8% polyacrylamide–urea gel followed by autoradiography. The RNA1 transcripts shown as controls were made from the rep promoter present in the plasmid DNA used as templates. Download figure Download PowerPoint Site-specific binding of HU is promoted by GalR binding and supercoiled DNA Affinity cleavage mapping by chemically converting HU into a nuclease gives information about the position of the reactive groups in HU in the DNA–protein complex (Lavoie and Chaconas, 1993; Lavoie et al., 1996). In order to analyze the presence of HU in the proposed nucleoprotein complex, we used HU modified with (EDTA-2-aminoethyl)-2-pyridyl disulfide–iron complex [EPD–Fe(III)] (Ebright et al., 1992; Ermácora et al., 1992) to locate the binding site of HU (Lavoie and Chaconas, 1993). The iron, Fe(III), chelated with EDTA is reduced by ascorbate to EDTA–Fe(II) which in turn reduces hydrogen peroxide, creating hydroxyl radicals (Tullius et al., 1987). Thus, the DNA regions near the amino acid residues derivatized by the reagent are cleaved through hydroxyl radical reactions indicating the binding of the modified HU in the vicinity. By converting HU into a 'nuclease' in this way, the HU binding site has been located in the transpososome recombination complex of bacteriophage Mu DNA (Lavoie and Chaconas, 1993; Lavoie et al., 1996). More than 60% of the HU molecules we derivatized contained one or more molecule of EPD–Fe(III) as judged by an electrophoretic analysis on an SDS–polyacrylamide gel. The 'HU-nuclease' preparations did not show any detectable reduction in its co-factor activity in the GalR-mediated repression of transcription in vitro (data not shown). Figure 2 shows the cleavage pattern of the two strands of DNA by HU-nuclease. The results clearly demonstrate HU binding to gal DNA at a specific region between the two operators. Although weak cleavages by HU-nuclease were observed in the absence or presence of the reducing agent Na-ascorbate, some of which could be artifacts of primer extension assay (lanes 1, 3, 6 and 8), the addition of GalR dramatically enhanced site-specific cleavages on both gal DNA strands at specific sites (lanes 4 and 9). Cleavage sites ranged from the nucleotide position −8 to +35 relative to the start point of transcription from the promoter P1. The site-specific cleavages were attenuated by the addition of inducer, D-galactose (lanes 5 and 10), clearly indicating that GalR binding to the operators stimulates HU binding to specific sites in gal. Since no significant cleavages were made by HU-nuclease prepared without the cross-linker, 2-iminothiolane, it argues against the possibility that a nuclease contamination of EPD–Fe(III) resulted in non-specific cleavages (lanes 2 and 7). This was also supported by hydroxyl radical DNA cleavage experiments which showed that HU did not create additional hypersensitive cleavage sites for DNA nicking reactions similar to that with HU-nuclease both in the presence or absence of GalR (data not shown). Incidentally, we have also observed that HU-nuclease enhances cleavages at the Rho-independent transcription terminator sequence (trpoC) present in the gal DNA (lanes 3–5). Such a sequence, which contains a dyad symmetry followed by a stretch of AT base pairs, very likely generates a cruciform structure that attracts HU. This is consistent with the previous reports that HU prefers to bind to distorted DNA. Figure 2.HU-nuclease footprinting on gal DNA. HU-nuclease used was made by coupling of HU with iron–EDTA in the presence (modified; lanes 1, 3–5, 6 and 8–10) or absence (unmodified; lanes 2 and 7) of a cross-linking agent 2-iminothiolane. Supercoiled gal DNA template (2 nM) was incubated with different combinations of GalR (40 nM), HU (80 nM) and D-galactose (10 mM), as indicated, under conditions of the in vitro transcription. After a hydroxyl radical reaction as described in Materials and methods, nicked DNA was purified and used as a template to synthesize complementary DNA strands with 32P-end labeled oligonucleotide primers. Dideoxy chain termination reactions were performed using wild type gal DNA and the same oligonucleotide primer (lanes G and C on bottom and top strands respectively). Nucleotide positions relative to the start site of the transcription from the P1 promoter are indicated on both sides. Closed and open vertical bars indicate the position of gal operators and transcription terminator respectively. Download figure Download PowerPoint HU has been described as an accessory factor that assists the formation of specific nucleoprotein complexes in many other DNA transactions (Johnson et al., 1986; Bramhill and Kornberg, 1988; Hodges-Garcia et al., 1989; Ogawa et al., 1989; Baker and Mizuuchi, 1992; Lavoie and Chaconas, 1994; Segall et al., 1994; Bètermier et al., 1995; Pérez-Martin and deLorenzo, 1995). In some of these cases, HU could be either displaced by high concentrations of salt or replaced by other proteins such as IHF or HMG1. We tested the stability of the GalR-induced DNA binding of HU to gal DNA by competition assays (Figure 3). Interestingly, the DNA cleavages caused by the EPD–Fe(III)-derivatized HU pre-bound to the DNA in the presence of GalR were not affected by the subsequent addition of a 5-fold excess of 'cold' HU before the Na-ascorbate treatment (lane 4), whereas pre-bound 'cold' HU inhibited significantly the cleavages by a 5-fold excess of derivatized HU added later (lane 3). The inhibition of intensities of cleavages in the latter case was quantified to be ∼75%. IHF and HMG1 did not compete effectively with the derivatized HU (lanes 5–8); the reduction of cleavages were up to 30% at specific spots. We have previously shown that IHF does not replace HU in repressing gal transcription (Aki et al., 1996). Thus, the GalR-dependent HU binding to gal is specific and relatively stable. GalR dimer is also specific for inducing site-specific binding of HU. The binding of homologous GalS (an isorepressor of GalR with 86% amino acid similarities) to OE and OI did not show the enhanced DNA cleavages by HU-nuclease. More interestingly, when wild type tetrameric LacI which generates DNA looping in gal, or dimeric LacIadi which does not, was used in HU binding experiments where gal operators were replaced by lac operators, neither protein supported HU binding. Apparently HU does not prefer or recognize a LacI-mediated DNA loop (see Discussion). Figure 3.Competition assays on HU-nuclease footprinting. The footprinting reaction was performed in the presence of GalR with cold HU (100 nM; lane 1) or iron–EDTA-derivatized HU (∼20 nM; indicated in box in lanes 2–8). For competition assays, cold HU (100 nM; lanes 3 and 4), IHF (100 nM; lanes 5 and 6), or HMG1 (300 nM; lanes 7 and 8) was added before (lanes 3, 5 and 7) or after (lanes 4, 6 and 8) the addition of derivatized HU and incubated for 10 min. The DNA cleavages by the addition of ascorbate were detected by the primer extension reactions as described in the legend of Figure 2. The result shown here is from the top strand of wild type gal DNA. Lane G shows the sequencing ladder obtained by dideoxy termination reaction with the same DNA template and primer. Download figure Download PowerPoint We also observed that HU binding to gal DNA did not occur when using a relaxed (topoisomerase I-treated) or a linearized (restriction enzyme-digested) gal DNA template (data not shown). This suggests either an energetic or a specific topological requirement for DNA in one of the steps leading to the assembly of HU and GalR on gal DNA. The GalR-dependent 'HU-nuclease' DNA cleavage sites, which were clearly observed, and the intensities with which they were cleaved on two gal DNA strands are diagrammed in Figure 4. Although the cleavage sites were distributed from position −8 to position +35, the major impact was in the region −5 to +23. The cleavage sites can be grouped approximately into four clusters. The four sets of cleavage sites overlapped three incomplete dyad symmetry sequences. Since the approximate centers of the four clusters of the cleavage sites were at 8–10 bp intervals, all of them would be more or less on one face of the DNA double helical cylinder (10.5 bp/turn) but somewhat distributed around the cylinder. The results were similar to that observed in Mu transpososome complex containing HU (Lavoie and Chaconas, 1993). We also note that the center of the entire region containing the cleavage sites in gal was at the nucleotide position +6.5 and not at the center (−5) of the two operators, i.e. the center of the proposed DNA loop. A nuclease cleavage experiment was performed without the use of 2-iminothiolane in which the α-subunit of the HU heterodimer became the only source of the nuclease activity because of a A43C substitution. In this experiment only the −12 to +6 segment of gal DNA showed HU cleavage patterns (Figure 4). These results strongly suggest that only one molecule of HU is responsible for all of the gal specific cleavages and the binding of the heterodimer is orientation specific. Since an HU heterodimer contacts two minor grooves in DNA through polypeptide loops, the center of gal DNA occupied by HU would be located at position +6.5 (Figure 4). Figure 4.Map of HU-nuclease cut sites observed in the presence of GalR. (Upper figure) Control region of the E.coli gal operon showing the location of the two promoters, P1 and P2, and two operators, OE and OI. (Lower figure) Helical representation of HU-nuclease cleavage sites aligned with nucleotide sequence of gal DNA (−15 to +39). Horizontal comb-like bars indicate incomplete dyad symmetric sequences. Intensities of the bands detected in lanes 4 and 9 in Figure 3 were quantified. Only strong cleavage sites with at least 40% intensities were considered. Large and small circles indicate the sites with 70–100% and 40–70% intensities of nuclease cleavages respectively. The marked segment below the horizontal double helix indicates the region −12 to +6 in contact with the HU α-subunit as discussed in the text. The filled circle shown in the double helix represents the center of the segment that is in contact with HU heterodimer. Download figure Download PowerPoint HU binding requires occupancy of both operators by GalR The repression of the two overlapping gal promoters both in vivo and in vitro requires GalR binding to both gal operators (Adhya and Miller, 1979; Irani et al., 1983; Haber and Adhya, 1988; Aki et al., 1996; Figure 1, lanes 3, 8 and 10). To test the contribution of the operators in site-specific HU binding, HU-nuclease cleavage experiments were conducted using operator mutant DNAs (Figure 5). It is clear that if one of the operators, OI, is deleted from the gal DNA, 'HU-nuclease' fails to show any site-specific gal DNA cleavage. This was true for DNA templates with two different OI deletions (line 1 versus lines 3 and 4). This and the following results suggest that the binding of GalR to both OE and OI is required to facilitate HU binding. HU binding, in turn, results in co-operative binding of GalR to OE and OI, presumably by an interaction between two DNA-bound GalRs as shown below. Figure 5.Effect of mutations or deletion of gal operator on HU-nuclease footprinting. DNA cleavages were detected using HU-nuclease as in Figure 3. Supercoiled DNA templates used were (from top to bottom) pSA509, pSA532, pSA-GD1, pSA-GD2, pSA512 and pSA510 (see Materials and methods). Closed and shaded boxes indicate gal and lac operators respectively. The results shown are for the top strand of DNA. Experiments that showed strong or weak cleavages are represented with ++ or + respectively. Download figure Download PowerPoint Although the binding of GalR to OE and OI by plasmid-borne operator titration experiments has been indicated to be co-operative in vivo (Irani et al., 1983; Haber and Adhya, 1988), in vitro binding of GalR to the two operators is non-co-operative (Brenowitz et al., 1990). Since site-specific HU binding to gal DNA is strictly dependent upon GalR interaction with both operators, we reinvestigated the co-operative binding of GalR using the 'HU-nuclease'-dependent DNA cleavage as a diagnosis of GalR co-operativity. We nevertheless observed significant site-specific HU-nuclease cleavages of gal DNA carrying one critical change in each half of the dyad symmetry at OE or OI (lines 2 and 5). We have previously shown that such mutations abolish its intrinsic binding to GalR as judged by DNase I protection assays (Majumdar and Adhya, 1986; Brenowitz et al., 1990). When both operator loci carried the mutations, the cleavages were no longer significant (line 6). These results clearly show that in the presence of HU, GalR binding to a wild type operator can rescue repressor binding to a mutant operator. Site-specific HU binding to gal is essential for such GalR co-operativity; the repressor did not bind to the mutant operators if HU was absent (data not shown). HU binding with GalR occupying two wild type operators is more stable than that with GalR occupying one wild type and the other mutant operator; HU binding in the former case is resistant to heparin challenge whereas in the latter it is not (data not shown). The simplest interpretation of such co-operative binding is an interaction between two GalR dimers in the presence of HU. Thus, an interaction of two DNA bound GalRs stabilizes site-specific binding of HU and vice versa. The nucleoprotein complex of GalR, HU and gal DNA prevents open complex formation We tested whether RNA polymerase binds to gal promoter in the presence of GalR and HU by DNase I protection analysis. DNA–protein complexes were formed by using a template DNA carrying a promoter mutation at P1 (P1−P2+) in the presence of heparin. The P1− mutation (a G→A transition at the nucleotide position −14) while abolishing any detectable P1 transcription, did not affect the action of GalR and HU in repression of gal transcription from the P2 promoter (Figure 1, lanes 11–14). DNase I experiments show the protection of the P2 promoter (−40 to +20) by RNA polymerase (Figure 6, lanes 1 and 5). The same protection pattern was obtained when GalR or HU was also present (lanes 6 and 7). RNA polymerase binding to P2 by DNase I protection assay was not observed in the presence of both GalR and HU (lane 8). Similar inhibition of RNA polymerase binding was also obtained by using wild type DNA (P1+P2+) (data not shown). Under such conditions, RNA polymerase, nevertheless, is able to bind and initiate transcription from RNA1 promoter present elsewhere in the plasmid DNA (Figure 1, lane 4). Thus, we conclude that the gal DNA–HU–GalR complex hinders heparin-resistant open complex formation by RNA polymerase at the gal promoters. If RNA polymerase binds to the promoter, such a complex is unstable under the conditions used. Interestingly, HU binding in the presence of GalR neither showed protection from nor generated hyper-reactive sites by DNase I treatment (lanes 4 and 8). Footprinting experiments probed by free iron–EDTA complex were also unable to detect HU binding (data not shown). Figure 6.DNase I protection of gal DNA. Supercoiled P1−P2+ gal template was incubated in the presence or absence of GalR, HU or RNA polymerase as indicated under conditions of the in vitro transcription except that dNTPs were omitted. After probing of the DNA–protein complex with DNase I, the nicked DNA was purified and used as a template for synthesizing complementary strands with 32P-end labeled oligonucleotide primers. Results shown are from the top strand of DNA. Dideoxy chain termination reactions were performed using wild type gal DNA and the same oligonucleotide primer (lanes G, A, T and C). Nucleotide positions relative to the start site of the transcription from the P1 promoter are indicated on the right. Closed vertical bars indicate the positions of gal operators. Download figure Download PowerPoint Discussion HU is an operon-specific transcriptional regulator It has been proposed previously that an interaction of two GalR dimers bound to spatially separated operators forming a DNA loop is a determinant of repression of the intervening promoters (Irani et al., 1983; Haber and Adhya, 1988). Indeed, DNA looping occurs frequently in multipartite operator systems for regulation of gene expression (Adhya, 1989). In an experimental gal system in which the two gal operators were replaced by lac operators, it has been demonstrated both in vivo and in vitro that Lac repressor (LacI) binding to the operators represses the initiation of transcriptions from the two gal promoters via DNA looping (Haber and Adhya, 1988; Mandal et al., 1990; Choy and Adhya, 1992). As expected, such an effect is abolished if a mutant LacI (LacIadi), which fails to tetramerize but interacts with lac operator normally, is used (Mandal et al., 1990; Alberti et al., 1991; Brenowitz et al., 1991; Choy and Adhya, 1992). GalR, like LacIadi, is a dimer in solution; it does not tetramerize under physiological conditions in vitro (Brenowitz et al., 1990). Furthermore, GalR, like LacIadi, represses transcription from P1 somewhat inefficiently and activates that from P2 (Choy and Adhya, 1992; Figure 1). In vivo, however, GalR, like LacI and unlike LacIadi, represses gal transcription from both P1 and P2 (Haber and Adhya, 1988; Mandal et al., 1990). We have demonstrated that the histone-like protein HU restores the ability of GalR to coordinately repress the gal promoters in vitro (Aki et al., 1996). The GalR and HU mediated repression of the two gal promoters requires both gal operators and is sensitive to the addition of inducer, D-galactose (Figure 1). HU failed to assist LacIadi in bringing about the concomitant repression of the gal promoters. The results of DNA cleavage by HU-nuclease showed that HU binds, or at least closely approaches, gal DNA in a site-specific way. The properties of site-specific stable binding of HU parallel the properties of HU in transcriptional repression—the requirement for binding of GalR to both operators, the requirement for supercoiled DNA and the sensitivity to the presence of inducer, D-galactose. The total dependency of site-specific HU binding for gal repression and its responsiveness to inducer shows for the first time that HU acts as an operon specific transcriptional regulator. Co-operative binding of HU, GalR and DNA looping Whereas HU has been shown to bind to 9 bp segments of DNA nonspecifically and transiently at micromolar concentrations (Broyles and Pettijohn, 1986; Bonnefoy et al., 1994), HU bound to −10 to +30 segment of gal DNA with an affinity in the nanomolar range (Figure 2). HU binding to gal is not only site-specific but also orientation-specific. The center of HU heterodimer binding is position +6.5 with the α subunit preferring the OE side on gal DNA. In spite of such orientation-specific binding, it is not clear whether HU binding to gal is sequence-specific. This region is AT-rich and contains three overlapping weak dyad symmetries. Such sequences have the potential for making a distorted structure when the DNA is supercoiled and thus becomes a target of HU. The site-specific stable binding of HU to gal DNA is totally dependent upon GalR binding to both operators, OE and OI. In turn, HU helps co-operative binding of GalR to OE and OI. A typical explanation of co-operative binding of proteins to multipartite DNA sites is protein–protein interactions between the co-operative partners (Hochschild and Ptashne, 1986). In this model, co-operative GalR binding is brought about by an interaction between OE- and OI-bound GalR molecules, resulting in the formation of a DNA loop. Loop formation in gal DNA has been independently confirmed by imaging GalR–HU–DNA complex by atomic force microscope (Lyubchenko et al., 1997). Architecture of GalR–HU–gal DNA complex We have considered different roles of HU in forming the GalR–HU–gal DNA complex responsible for repression of transcription (Aki et al., 1996). (i) HU molecules wrap solenoidally around the promoter DNA. White et al. (1989) have proposed such a structure in which several HU dimers bind to DNA side by side. (ii) HU helps DNA looping by acting as a DNA bender, as a protein adaptor, or both. As a bender, it binds to a DNA site between OE and OI and stabilizes a transient interaction between two operator-bound GalR dimers. As an adaptor, HU binds simultaneously to OE- and OI-bound GalR, which are otherwise incapable of interaction. The architecture of GalR and HU binding to gal DNA complex is somewhat similar to the architecture of site-specific recombination complexes in bacteriophage Mu and λ (Goodman et al., 1992; Lavoie and Chaconas, 1994). In the case of MuA–HU–DNA transpososome structure, Lavoie and Chaconas (1993, 1994) and Lavoie et al. (1996) have shown that one HU heterodimer binds around the center of the two MuA binding sites, creating a footprint of ∼30 bp because of HU-induced DNA bending. HU binding and bending of the intervening DNA is proposed to stabilize a weak interaction between the two MuA monomers. A similar architecture has also been described in the formation of the intasome complex in bacteriophage λ, in which a bivalent Int protein contacts two distal DNA sites with an IHF molecule protein binding in the intervening region and bendin
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