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© 1997 Oxford University Press 3490-3496

Contacts between Bacillus subtilis catabolite regulatory protein CcpA and amyO target site

Contacts between Bacillus subtilis catabolite regulatory protein CcpA and amyO target site Jeong-Ho Kim and Glenn H. Chambliss*

Department of Bacteriology, University of Wisconsin-Madison, E. B. Fred Hall, Madison, WI 53706, USA

Received May 2, 1997; Revised and Accepted July 11, 1997

ABSTRACT

Catabolite control protein A (CcpA) is a global regulatory protein involved in catabolite repression and glucose activation in Gram-positive bacteria. cis-Acting DNA sequences, catabolite response elements (cres), involved in this regulatory system contain a 14 base pair (bp) region of dyad symmetry. CcpA, a repressor of the LacI family, has been shown to bind specifically to cres. To better understand cre recognition by CcpA, we have focused on the interaction between CcpA and the amyEcre, called amyO, which is located at the transcription start site of the [alpha]-amylase gene. DNA-protein complexes were probed with dimethylsulfate (DMS) and N-ethylnitrosourea (EtNU) to identify guanines and phosphates that participate in complex formation. Interaction between amyO and CcpA visualized through methylation protection and interference showed that CcpA contacts guanine residues at the outer bounds of amyO with higher affinity than near the dyad axis. From ethylation interference studies, it was found that CcpA contacts three phosphate groups at each end of amyO, and one or two phosphate groups near the dyad axis. Exonuclease III protection revealed that CcpA protects a 26 bp region centered around the dyad axis of amyO. The isolated N-terminal fragment still specifically bound to the sequence resembling the half sites of the amyO sequence. Considering these findings and the helical structure of B-DNA, our results suggest that each of the two monomers of the CcpA molecule contact the major groove in each half of the region of dyad symmetry and that the contacts are on the same face of the DNA helix, which is typical of bacterial repressor-operator interactions. However, the absence of strong contacts near the dyad axis by CcpA is in contrast to the situation with the gal repressor, another member of the LacI family of repressors.

INTRODUCTION

Expression of Bacillus subtilis [alpha]-amylase gene (amyE) is dramatically repressed by the presence of readily metabolized carbon sources in the culture medium. This phenomenon is called catabolite repression because of the similarity to the situation observed in enteric bacteria such as Escherichia coli (1 ,2 ). In B.subtilis genetic studies of amyE expression have revealed that at least two regulatory elements, a cis-acting DNA sequence, designated amyO,and a trans-acting factor, CcpA, are involved in catabolite repression (3 -5 ). The amyO sequence, the first catabolite repression operator identified (6 ),spans the transcription start site of the amyE and exhibits partial dyad symmetry (4 ). A cis-acting mutation, gra10, which alters position +5 (G to A), and other mutations of amyO relieved catabolite repression of [alpha]-amylase synthesis (4 ,6 ). Based on genetic analysis, the deduced consensus sequence of the 14 bp amyO is as followed; TGWAANC*GNTNWCA. The most critical bases are underlined, N is any base, W indicates adenine or thymine, and the asterisk denotes the axis of dyad symmetry (6 ). amyO has sequence identity to lacO (79%) and consensus galO (92%). Interestingly, sequences similar to amyO, called cres, have been found in many genes and operons of Bacillus (7 ). Mutations in cres relieved catabolite repression of specific genes in B.subtilis (8 -10 ) and Bacillus megaterium (11 ), suggesting that cres are regulation targets of a repressor protein.

CcpA is a trans-acting gene product affecting catabolite repression of [alpha]-amylase gene expression (5 ). Analysis of the deduced amino acid sequence suggested that CcpA is a procaryotic repressor belonging to the LacI-GalR family. These repressors contain a helix-turn-helix DNA binding domain, an effector binding site, and a dimerization domain (5 ,12 ). Subsequent biochemical studies have revealed that CcpA is a dimer composed of two 38 kDa monomers and binds specifically to wild type amyO, but not to mutant amyO (13 ). A specific CcpA binding site spanning amyO was identified by DNase I protection (13 ). CcpA and cres are required for regulation of not only [alpha]-amylase synthesis, but also of genes or operons which are subject to catabolite repression (9 ,10 ,14 ,15 ) or glucose activation (16 ), suggesting the two elements play a pivotal role in the regulation of central metabolism in Gram-positive bacteria.

As an initial step in generating an understanding, at the molecular level, of amyO-CcpA recognition, we probed the interaction between CcpA and amyO using protection and modification interference techniques with small chemicals: dimethylsulfate (DMS) for purine base specific interactions; and N-ethylnitrosourea (EtNU) for phosphate backbone interactions. In addition, Exonuclease III was used to identify the boundaries of the CcpA binding region. To more precisely define the DNA-binding domain of CcpA, we examined the amyO-binding ability of the N-terminal fragment of the CcpA containing a helix-turn-helix motif.

MATERIALS AND METHODS

Preparation and labelling of DNA

The amyO region (-27 to +27) from pAMYO (13 ) and the whole regulatory region (amyR) encompassing the amyE promoter and amyO (-111 to +96) from pAMR were used as probe DNA fragments for CcpA and truncated CcpA. Plasmid pAMR was constructed by amplifying the 207 bp of the amyR fragment by polymerase chain reaction (PCR) using Vent polymerase (New England Biolab Co.) and cloning the amplified fragment into the HincII site of plasmid pUC18. The primers used for PCR were -111GGAAAGCGAGGGAAGC G-95 and +96GAAGACTTACTTCG GAGTCA+77.

Isolation and 3'-end labelling of the amyO and amyR fragments from the plasmids were as described previously (13 ). For 5'-end labelling of the amyR fragment, pAMR was firstly digested with BamHI for labelling the coding strand or HindIII for labelling the template strand. After dephosphorylation with shrimp alkaline phosphatase (United State Biochemicals Co.), the plasmids were redigested at the secondary restriction sites, HindIII site for the coding strand and BamHI site for the template strand to liberate the amyR fragment. The fragments were then 5" end-labelled with [[gamma]-32P]ATP (3000 Ci/mmol, Amersham Co.) using T4 polynucleotide kinase, separated in a 5% non-denaturing polyacrylamide gel, and the amyR fragment was isolated by electroelution.

Dimethylsulfate protection

Formation of amyO-CcpA complex was carried out as described previously (13 ), except that a 5-fold excess of CcpA was used in order to stabilize the DNA-proteincomplex. Methylation of DNA with DMS (Aldrich Co.) was performed as described by Maxam and Gilbert (17 ) with minor modifications. For DNA methylation, 0.2% (v/v, final concentration) of DMS was used to treat 30 [mu]l naked DNA (1 pmol) or DNA-protein complex. After incubation at 20oC for 1 min, the reaction was stopped by adding 15 [mu]l DMS stop solution (1.5 M Na-acetate, pH 7.0, 1.0 M [beta]-mercaptoethanol, 15 mM EDTA, 100 [mu]g/ml calf thymus DNA) and 120 [mu]l of cold ethanol. Cleavage at the methylated bases in both the bound and free DNA fragment was accomplished by incubation in 50 [mu]l of 10% (v/v) piperdine and 10 mM EDTA at 905C for 30 min. After lyophilization, the DNA samples were resolved in 10% polyacrylamide sequencing gels containing 8 M urea. DNA band intensities were measured using a PhosphorImager with imageQuaNT software (Molecular Dynamics Co.) from the autoradiographed gel and protection was quantified by normalizing the intensities of the affected bands to that of an unaffected band located outside of amyO sequence. The extent of protection was shown as the ratio of band intensities in the absence (C) versus the presence of CcpA (P). As the P/C ratio is given as a logarithm, negative values indicate protection and positive values reflect enhancement of sensitivity.

Dimethylsulfate interference

DMS interference experiments were carried out in a manner similar to that of Siebenlist and Gilbert (18 ). The DNA fragment was methylated in 200 [mu]l 50 mM Na-cacodylate, pH 8.0, 10 mM MgCl2, 0.1 mM EDTA, and 50 mM NaCl by treating with 1% (v/v, final concentration) DMS at 205C for 1 min. After stopping the reaction by the addition of 50 [mu]l DMS stop solution and precipitating the DNA with 3 vol of cold ethanol, the DNA samples were redissolved in distilled water. CcpA was then incubated with the premethylated amyO fragment for the complex formation. Free amyO fragments and the amyO-protein complexes were resolved in 5% non-denaturing gels, electroeluted separately, and precipitated. Cleavage at the methylated bases and separation of the DNA samples were performed as described for methylation protection.

Ethylation interference

The amyO fragment was ethylated with ethanol-saturated EtNU (Aldrich Co.) at 505C for 60 min as described by Siebenlist and Gilbert (18 ). After CcpA was added to the previously ethylated DNA, free DNA fractions were separated from the DNA-protein complexes in a non-denaturing gel. Recovery of the bound and free DNA and DNA precipitation procedures were done as described in the DMS interference experiment. To cleave ethylated phosphates, 2.5 [mu]l 1 M NaOH was added to 15 [mu]l DNA redissolved in 10 mM phosphate buffer, pH 7.0 and 1 mM EDTA, and then incubated at 905C for 30 min. After heating, the samples were neutralized with acetic acid, precipitated, and analyzed in DNA sequencing gels.

Exonuclease III protection

Experiments were carried out as described by Wu (19 ) with the following modifications. The 5" end-labelled amyO fragment was incubated in an assay mixture of 30 [mu]l containing 2.5 [mu]g poly(dI@dC)-poly(dI@dC) (Pharmacia), 1 [mu]g bovine serum albumin, binding buffer (20 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 1 mM dithiothreitol (DTT), 1 mM EDTA, 5% glycerol, 1 mM NaCl), and purified CcpA (0.5 [mu]M) at room temperature for 15 min. After formation of DNA-protein complex, 300 U Exonuclease III (Promega Co.) were added to the mixture for 5 min. The reaction was terminated by the addition of 60 [mu]l DNase I stop solution (10 mM EDTA and 100 [mu]g/ml of yeast tRNA) and 1% sodium dodecyl sulfate (SDS). The DNA samples were resolved in 8% polyacrylamide gels containing 8 M urea.

Tryptic digestion of CcpA

To establish optimum digestion conditions, CcpA was treated in a buffer containing 0.01 M CaCl2, 0.3 M NaCl and TGED solution (50 mM Tris-HCl, pH 8.0, 10% glycerol, 0.1 mM EDTA, and 1 mM DTT) with various concentrations of trypsin, treated previously with L-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK, United State Biochemical Co.), for varying lengths of time. After digestion, the reaction was stopped by adding 0.2 mM diisopropylfluorophosphate. The tryptic digests of CcpA were subjected to gel filtration chromatography on Sephacryl S-100-HR (Sigma Co.) column (1.5 * 100 cm). Purity and molecular size of the digestion products was judged by electrophoresis on SDS-20% polyacrylamide gel. DNA-binding ability of the peptides was monitored by gel retardation assay as described previously (13 ). N-terminal sequencing of the small fragment was performed at the protein/DNA facilities in the UW-Milwaukee.


Figure 1. Methylation protection of amyO by CcpA. The 3"-end labelled amyO (C) and amyO-CcpA complex (P) were treated with DMS (0.2%, v/v) and methylated bases were cleaved with 50 [mu]l of piperdine (10%, v/v) and 10 mM EDTA. After electrophoresis of the DNA, electrophoretic patterns in the coding strand (A) and in the template strand (B) were autoradiographed. amyO (-3 to +11) is shown enclosed in a box for both strands. A/G represents A+G ladder of the amyO fragment.

RESULTS AND DISCUSSION

Purine-CcpA contacts

DMS predominantly methylates the N-7 atoms of guanines in the major groove of B-DNA, and about five times more slowly, the N-3 atoms of adenines in the minor groove (20 ). amyO-CcpA interactions were reflected either as changes in the DMS sensitivity caused by bound CcpA in methylation protection, or as selective binding of CcpA to the amyO fragments methylated on residues dispensible for CcpA binding in methylation interference experiments. CcpA protected all guanine residues within amyO, corresponding to positions -2, +3 and +5 in the coding strand, and +4 and +10 in the template strand (Fig. 1 ). The degree of protection on the guanine residues, however, depended on the position of the residue within the region of dyad symmetry. Guanine residues at -2 (coding strand) and +10 (template strand) which are on each end of amyO were strongly protected, whereas guanine residues at +3 and +5 (coding strand) and +4 (template strand) which are near the dyad axis were less well protected. Adenine residues were not detected in these experiments due to the low concentration of DMS used. The binding affinity of CcpA at guanine residues, given as logP/C ratio, shows strong contact only at the outermost regions of amyO (Fig. 2 ). In CcpA titration assays using DMS protection, the guanine residues at -2 and +10 were very sensitive to protection as CcpA concentration was increased, but those near the dyad axis were not (data not shown). Even though the guanine residues at -2 in the coding strand and +10 in the template strand are located the same distance from the dyad axis, the strength of the guanine-CcpA contacts were not equal, that is, CcpA contacted +10 of the right half of the sequence with ~2.5-fold higher affinity than to -2 of the left half. This is comparable with LacI exhibiting preferential binding affinity for the left half of the natural lacO (21 ).


Figure 2.Quantitative analysis of DMS protection of amyO by CcpA. The degrees of protection were quantified by measuring band intensities using a densitometric scanner (PhosphorImager) from the original data shown in Figure 1. The extents of protection are given as a logarithm, log(P/C), in which P and C indicate band intensities at the depicted sites in the presence (P) and absence of CcpA (C). Therefore, various negative values shown as lengths of black bars indicate degrees of protection at each guanine residue within amyO (box).

In methylation interference experiments, amyO was methylated and then exposed to CcpA. Methylation of the DNA was partial with approximately one methylated base per DNA molecule. CcpA would be expected to only bind to amyO methylated at sites that are not critical for CcpA contact. The absence of bands in the CcpA bound fraction and their presence in the free fraction indicate critical CcpA binding sites (Fig. 3 ). As would be predicted from the results of the methylation protection experiments, guanine residues at -2 and +5 in the coding strand and at +10 in the template strand were detected with strong band intensity, while those at +3 in the coding strand and +4 in the template strand were detected with less band intensity. Interference probing with DMS identifies guanine residues which are indispensible for DNA-protein complex formation; therefore, the guanine residues at -2 and +5 in coding strand and at +4 and +10 in the template strand, which are symmetrically positioned in the DNA sequence, are crucial for CcpA binding. This observation is noteworthy in that the CcpA contacts at positions -2 and +5 in the coding strand are the same distance apart as are the contacts at +4 and +10 in the template strand, suggesting symmetrical binding of CcpA to the two amyO half sites.


Figure 3.Methylation interference of amyO by CcpA. End-labelled amyO fragments of both strands, the coding strand (A) and the template strand (B), were methylated with DMS and then incubated with CcpA. After formation of amyO-CcpA complexes, bound (B) and free (F) DNA fractions were separately isolated from a non-denaturing gel after separation by gel retardation (13). Cleaving of the methylated bases, resolving of the amyO fragments in a sequencing gel, and autoradiographs were done as described in Materials and Methods.


Figure 4.Ethylation interference of amyO by CcpA.The amyO fragments of both strands, the coding strand (A)and the template strand (B), were pre-ethylated with ethanol-saturated EtNU and then incubated with CcpA. As in the DMS interference experiment, the two sets of amyO fragments, bound (B) and free (F), were separately isolated and the ethylated phosphates were cleaved. After resolving the amyO fragment in a sequencing gel, the ethylation patterns were analyzed from the autoradiograph of the gel. Ethylation of the phosphates marked by arrows interferes with CcpA binding to the amyO fragment. As a control, ethylated amyO fragment (C) was also resolved in the gel. A/G and C/T represent sequencing ladders of A+G and C+T, respectively.


Figure 5.Comparison of phosphate-repressor contacts between LacI, GalR, and CcpA and its operators. The contact sites of LacI (22) and GalR (23) were redrawn from the referenced papers and those of CcpA were drawn from Figure 4. Arrows indicate phosphate contacts in each DNA-protein complex and boxes indicate each operator.

Phosphate backbone-CcpA contacts

To identify CcpA-phosphate contact sites, the ethylation interference method was utilized. In this procedure, CcpA was bound to amyO fragment previously ethylated with EtNU. As in methylation interference, bands with enhanced intensity in the free fraction and reduced intensity in the bound fraction indicate phosphates at which ethylation of amyO inhibits CcpA binding (Fig. 4 ). The results indicated that the four phosphates located at positions -2, -3, -4, and +5 in the coding strand and the five phosphates located at positions +3, +4, +10, +11, and +12 in the template strand are required for CcpA binding, showing symmetrical binding of CcpA centered around the dyad axis. As is true with LacI and GalR binding to their respective operators (22 ,23 ), CcpA contacts phosphates beyond the 14 bp consensus sequence in both strands of amyO. The phosphate groups at the ends (-2 in the coding strand and +10 in the template strand) and those near the dyad axis (+5 in the coding strand and +4 in the template strand) of the amyO sequence that interfere with CcpA binding when ethylated are separated by five non-interfering ethylated phosphates. This suggests that each subunit in the dimeric CcpA molecule interacts with a phosphate at the outer end of an amyO half site and with a second phosphate at the internal end of that half site. Between these two contact points there are five phosphates at which ethylation does not interfere with CcpA binding. The distance between the interfering ethylated phosphate groups in a given DNA strand is a half turn of the helix. This distance is 2 bp longer than that found with GalR binding to its operator (23 ). Phosphates participating in the CcpA contacts around the dyad axis of amyO are one or two per strand whereas GalR contacts three phosphate groups near the dyad axis in each strand of galO (Fig. 5 ). These observations would suggest that CcpA does not bind as tightly near the dyad axis as does GalR. Since ethylation interference detects only phosphate groups which are critical for complex formation rather than accessibility of individual phosphates, the displayed phosphates are presumed to play a substantial role in protein-DNA contact. On this basis, it is clear that CcpA does not bind near the dyad axis nearly as strongly as it does to the ends of amyO. This binding pattern is in direct agreement with the results obtained from examination of the guanine-CcpA contacts.

Escherichia coli GalR has 31% amino acid identity to CcpA and, consensus galO exhibits 92% sequence identity to amyO (5 ). As shown by DNase I footprints, GalR specifically binds to amyO and does so with ~100-fold higher affinity than does CcpA (data not shown). DMS footprinting showed that GalR binds tightly to all guanine residues within amyO including those at positions +3, +5 and -2 in the coding strand (data not shown), while CcpA binding at positions +3 and +5 was barely detectable (Fig. 2 ). These results suggest that CcpA and GalR complex with amyO differently. The CcpA binding pattern appears more comparable with the way phage 434 repressor binds its specific target, OR1 of the 434 operator (5"ACAAGAA*AGTTTGT3", * indicates the dyad axis and regions of great sequence similarity to amyO are underlined. Note the opposite orientation with regard to amyO). The 434 repressor does not contact directly the center of the operator, but the inner residues are believed to be important for bend formation upon repressor binding (24 ).

Mapping of boundaries of CcpA protection region


Figure 6.Mapping of CcpA protection region by Exonuclease III. The enzyme digested 5" end-labelled amyR fragment of the coding strand (A) and template strand (B) until it was blocked by bound CcpA (P). CcpA binding blocked Exonuclease III digestion at +17 in the coding strand and -9 in the template strand, respectively. DNA samples were resolved in an 8% polyacrylamide gel containing 8 M urea. A/G and C/T represent sequencing ladders of A+G and C+T, respectively.

Although DNase I protection revealed that CcpA binds to amyO, the exact boundaries of the protected region were not clear because DNase I digestion sites are lacking at the borders of amyO (13 ). To more clearly define the region covered by CcpA, we used Exonuclease III protection for its greater resolution of the protection boundaries (19 ). Since the 3" to 5" exonuclease would remove mononucleotides from each 3" end of an amyO containing DNA fragment in a processive way, it would digest free DNA until its action was blocked by CcpA bound to amyO, thus leaving double-stranded DNA only in that region where CcpA is bound (Fig. 6 ). CcpA binding blocked Exonuclease III digestion at positions +17 in the coding strand and -9 in the template strand, indicating that a region of 26 bp centered around amyO is covered by CcpA. The borders of the CcpA binding region determined in this manner are in good agreement to those deduced from DNase I protection (13 ).

DNA binding activity of N-terminal fragment of CcpA


Figure 7.Isolation and DNA-binding activity of N-terminal fragment of CcpA from trypsin digestion. (A) Tryptic digestion of CcpA. CcpA (20 mg) was incubated with 2 ng of TPCK-treated trypsin for various times (0-40 min) in reaction buffer containing CaCl2. After stopping the digestion reactions, the reaction mixtures were immediately electrophoresed in a 20% SDS-polyacrylamide gel. Molecular weight markers were ovalbumin (43 kDa), carbonic anhydrase (29 kDa), [beta]-lactoalbumin (18.4 kDa), lysozyme (14.3 kDa), bovine trypsin inhibitor (6.2 kDa), insulin ([beta]-chain, 3.4 kDa), and insulin ([alpha]-chain, 2.3 kDa). (B) Gel retardation assay of the N-terminal (NF) and large fragment (core) of CcpA. amyO fragment was incubated with the core (0.5 [mu]M) and N-terminal fragment of CcpA (from left to right ; 0.25, 0.5, 1.0, and 2.0 [mu]M) in a gel retardation buffer (13) and the reaction mixtures were run in a 5% non-denaturing polyacrylamide gel. amyO binding activity of CcpA (intact CcpA, 0.5 [mu]M) was also tested with the tryptic digests.


Figure 8.(A) Comparison of DNA binding specificity between the intact (I) and N-terminal fragment (NF) of CcpA. A volume of 0.2 [mu]M of each type of CcpA was used for amyO binding. After the whole regulatory region of amyE (207 bp fragment) was excised and labelled at 3"-end in the coding strand, the fragment was complexed with intact and NF of CcpA. (B) Unequal binding affinity of NF for each half of amyO in the template strand. Footprinting titration of amyO fragment with NF was carried out using DNase I protection without (C) and with NF (from left to right; 15, 30, 60, 120, 240, and 360 nM). For DNase I protection, 2 U DNase I were used for 30 [mu]l reaction vol for 20 s at room temperature as described previously (13). For quantification of the protection, +9T and +2T were used as diagnostic bands for the left and right half sequence, respectively, and +21T was used as a reference. The left and right halves of the amyO sequence are shown on the side of the figure.

The recognition helix of the helix-turn-helix motif in DNA-binding proteins recognizes its specifc DNA sequence. It has been demonstrated that the headpiece of lac repressor can specifically bind to the lac operator and repress [beta]-galactosidase production in vitro and in vivo (20 ,25 ). To determine if a similar DNA binding headpiece could be generated from CcpA which shares structural similarity with LacI, CcpA was subjected to digestion with trypsin. After limited digestion, two CcpA fragments of 4-5 kDa and 33-34 kDa were generated (Fig. 7 A). The presumed cleavage sites to generate the small fragment were R44, R48 or R54. Position R54 (R) is located in what is thought to correspond to a hinge region devoid of secondary structure. This region would be expected to be a preferred site for the trypsin digestion of CcpA as was the case with LacI (26 ) and PurR (27 ). The small fragment generated by trypsin digestion bound to amyO, whereas the large fragment of CcpA did not, indicating that the DNA binding domain is contained within the small fragment (Fig. 7 B). From sequencing the first 12 N-terminal amino acids, it turned out that the small peptide is the N-terminal fragment containing the DNA binding helix-turn-helix motif found in the CcpA sequence (5 ). When tested with the whole regulatory region of amyE given from the plasmid pAMR, the N-terminal fragment recognized not only the full amyO sequence, but also sequences similar to the amyO half site wherever they occurred in the DNA (Fig. 8 A). The N-terminal fragment bound to each half of amyO with a different affinity, showing a 10-fold higher preference for the left half than for the right half(Fig. 8 B). This is in contrast to that of intact CcpA which preferred the right half of amyO with 2.5-fold higher affinity than for the left half, based on DMS protection. One of the reasons that intact CcpA may bind amyO differently than does the N-terminal fragment of CcpA is that in the intact dimeric form, the orientation of the recognition helices of the DNA binding domains of the two monomers of CcpA are constrained in relation to each other in the binding to amyO (28 ). This is not the case with the N-terminal fragments, each of which presumably would be free to bind amyO in the highest affinity orientation. In the gel retardation assay, greater gel shifts were observed as the concentrations of the N-terminal fragment were increased, presumably because more than one peptide would bind to the amyO-containing DNA fragment. This supposition was corroborated by the DMS protection assay in which the N-terminal fragment protected all the guanine residues within amyO to almost the same extent, showing a remarkably different protection pattern from that shown by intact CcpA (data not shown). Therefore, these results suggest that in addition to the helix-turn-helix domain, regions within the core of CcpA, possibly those responsible for dimerization, are required for specific interaction of CcpA with the symmetrical dyad of amyO and not with half site sequences.


Figure 9. Summary of the various footprints (upper) and proposed model for CcpA binding to the amyO helix (lower). The CcpA binding region identified by DNase I protection (13) was confirmed by Exonuclease III mapping (opened arrow). Darkness of the circles at the guanine residues indicate the degree of the CcpA binding affinity. Phosphate contacts are indicated by dark arrows. The 14 bp amyO sequence is shown enclosed in a box and base positions relative to the transcription start site are indicated.

In summary, DNA binding proteins recognize specific DNA sequences through direct contacts between amino acid side chains of the protein and the exposed edges of the cognate base pairs, mainly in the major groove of B-DNA. In the model that has been proposed for operator binding by repressors of the LacI family, two recognition helices of the helix-turn-helix motif of dimeric DNA binding proteins are placed across the major groove of DNA centered about a region of dyad symmetry (29 ,30 ). Our results strongly indicate that each of the two monomers of the dimeric CcpA molecule contact the major groove at two sites on the same face of the amyO helix (Fig. 9 ), suggesting that the overall amyO-CcpAbinding pattern conforms to that of a typical bacterial repressor-operator interaction (31 -36 ). However, substantial close contacts in the central region of the amyO were not detected, which may be important in the mechanism of action of CcpA. Mutations at positions -2, +5, and +10, which were identified as crucial residues for CcpA binding in this study, disrupted the repression of [alpha]-amylase expression when glucose was present in the medium (6 ). Mutation at position +4 also greatly disrupted catabolite repression as much as mutations at -2, +5, and +10; however, CcpA contact at that position (+4 G), which is located in the center of amyO was barely detectable (Fig. 2 ). These results could suggest the requirement for a conformational change in molecular structure of CcpA by an effector molecule(s) so as to allow CcpA to bind the central region of amyO including position +4 with high affinity and thereby repress transcription from the [alpha]-amylase promoter.

ACKNOWLEDGEMENTS

This work was supported in part by the Department of Bacteriology, College of Agricultural and Life Sciences at UW-Madison and by National Institutes of Health grant GM 34324. The authors would like to thank Drs Michael J. Weickert and Sankar Adhya for their gift of purified GalR protein.

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*To whom correspondence should be addressed. Tel: +1 608 263 5058; Fax: +1 608 262 9865; Email: ghchambl@facstaff.wisc.edu
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