INTRODUCTION

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The presence of multiple activators permits a promoter to respond to multiple environmental cues. The way in which several activators work together is an important question in the study of transcription regulation. The araBAD promoter in Escherichia coli is regulated by two transcription factors, AraC and catabolite gene activator protein (CAP) (13). The main activator protein, AraC, binds to two direct-repeat half-sites that partially overlap the 35 region of the promoter (8, 32). The second activator, CAP, has a binding site centered at position 93.5 (15). The mechanisms of transcription activation by these two proteins have been studied extensively (5, 10, 32, 38).

AraC protein is composed of a dimerization domain and a DNA binding domain (7). In the absence of arabinose, the dimeric AraC protein binds to the upstream araO₂ half-site and the downstream araI₁ half-site and forms a DNA loop to repress transcription (9, 26) (Fig. 1). The presence of arabinose induces conformational changes in AraC that lead it to bind to two adjacent half-sites, araI₁ and araI₂ (24, 26). When bound at araI₁ and araI₂, AraC helps RNA polymerase to bind to the araBAD promoter, p_BAD, and also accelerates open-complex formation (38).

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Regulation of ara pBAD. Upon induction of arabinose, AraC releases the upstream araO2 half-site and binds to the downstream araI1-araI2 site. From this position, AraC activates transcription with the aid of the CAP.

CAP is the sole activator at two classes of simple E. coli promoters. At class I promoters, where CAP binds at position 61.5 or further upstream, amino acids 156 to 164 of CAP have been shown to be essential for transcription activation (2, 12, 40). This activating region, activating region 1 (AR1), directly contacts the alpha subunit of RNA polymerase (10, 19). At class II promoters, where the CAP binding site is centered at position 41.5, AR1 and amino acids 19, 21, and 101, which constitute activating region 2 (AR2), were both found to interact with the alpha subunit of RNA polymerase (5, 29). Also, amino acids 52 to 58, a region known as activating region 3 (AR3), lie close to the sigma subunit of RNA polymerase (5, 21, 37). In wild-type CAP, AR3 plays little or no role in transcription activation; however, substitution of amino acid 52 can result in increased transcription activation, presumably by creating a new, nonnative interaction between AR3 and sigma (5).

The role of CAP in systems with multiple activators has also been studied. At some promoters where another activator protein is bound between CAP and RNA polymerase, the AR1 region of the upstream CAP has been shown to be involved in transcription activation (6, 23). Apparently, CAP can still contact RNA polymerase even though the two are separated by the third protein. CAP may also act as a structural protein by bending DNA about 90°. It has been argued that this sharp bend may facilitate the binding of upstream DNA to the backside of RNA polymerase (4). CAP-induced bending has been shown to modulate the location of binding of the primary activator MalT to trigger transcription activation in the malK promoter (33).

CAP activates ara p_BAD in at least two ways. Previous studies have established that one role of CAP is to break the loop formed by AraC in the absence of arabinose, and thus CAP "activates" transcription by relieving repression (27). CAP also activates in a loop-independent way (15, 25, 36). When the upstream AraC half-site araO₂ was deleted to prevent DNA looping, CAP still substantially activated p_BAD (15). Although CAP activation requires the presence of AraC protein, sensitive in vitro studies did not detect any cooperative binding between CAP and AraC (16, 37a), and in a minicircle assay, CAP actually slightly destabilized AraC binding (27).

The loop-independent activation by CAP at ara p_BAD can be explained if CAP directly contacts RNA polymerase. A possible determinant on CAP for this interaction is AR1, since it has been shown to contact RNA polymerase at a large number of promoters. A previous study with an AR1 mutation, H159L, of CAP however, did not detect any effect at p_BAD (37), even though this mutation strongly disrupted the transcription activation of CAP at both class I and class II promoters. This finding left it unclear how CAP stimulates ara p_BAD.

In this study, we first established that CAP activates the p_BAD promoter when DNA looping is impossible. We also showed in in vivo experiments that CAP does not activate transcription by stabilizing AraC binding to the promoter. We then screened for CAP mutants defective in transcription activation and found mutants with amino acid changes in AR1 and in a region overlapping but not coincident with AR3. Our results suggest that CAP directly interacts with RNA polymerase to activate transcription at the araBAD promoter.

	MATERIALS AND METHODS

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General methods, culture media, and conditions. General methods were used as described previously (34). For the -galactosidase assay and in vivo footprinting, cells were grown in minimal medium, which includes M10 salts, 0.4% glycerol, 10 µg of thiamine per ml, 40 µg of leucine per ml, 0.2% Casamino Acids, and, when added, 0.2% arabinose or L-rhamnose. The -galactosidase assay was performed as described previously (11). For screening CAP mutants, the indicator plates contained 25.5 mg of antibiotic medium 2 (Difco) per ml, 50 µg of tetrazolium per ml, and 1% lactose.

Strains and plasmids. Strains XE64.2 (crp39 strA thi) and XE82 (crp39 strA thi lacPUV5-O^CAP) were provided by R. H. Ebright (40). To assay promoter activity in an AraC background, we used SH321 (araC-leu1022, araB⁺A⁺D⁺ lac74 galK Str^r), and for the AraC⁺ background, we used SH322 [ara(CBAD)⁺ leu lac74 galK Str^r] (14).

In vivo DMS footprinting. Cells (10 ml) were grown at 37°C in 125-ml flasks to an optical density at 550 nm of 0.8 to 1.0. Dimethyl sulfate (DMS) (10 µl) was directly added to the flask, and the cell culture was shaken for 2 min at 37°C. The culture was transferred to an ice-cold centrifuge tube and centrifuged at 1,000 × g for 5 min at 4°C. Plasmid DNA was isolated from the cell pellet and dissolved in 70 µl of distilled H₂O dH₂O plus 10 µl of piperidine. The solution was incubated at 90°C for 30 min, extracted with 1 ml of 1-butanol, and centrifuged at 15,000 × g for 15 min. The plasmid pellet was rinsed with 70% ethanol, dried, and resuspended in 15 µl of distilled H₂O. A PCR amplification was carried out in a 10-µl reaction mixture with only one ³²P-labeled oligonucleotide primer. For p10, the primer was 5'-AATAGGCGTATCACGAGGCCC-3'. For p10+CAP, the primer was 5'-TTATTTGCACGGCGTCACACTTT-3'. The reaction mixture contained 7 µl of piperidine-treated plasmid, 0.5 U of Taq DNA polymerase, 3 ng of ³²P-labeled oligonucleotide primer, 10 µM dATP, 10 µM dCTP, 10 µM dTTP, 20 µM dGTP, 10 mM MgCl₂, and 50 mM Tris-HCl (pH 9). The cycle parameters were 94°C for 1 min, 60°C for 1 min, and 65°C for 1 min, repeated for 29 cycles. The reaction products were run on a 6% denaturing polyacrylamide sequencing gel.

Random mutagenesis and screening of CAP. The DNA fragment containing the CAP gene was amplified by PCR with primers 5'-TCATCCGCCAAAACAGCC-3' and 5'-AATTAATCATCCGGCTCG-3'. Mutations were generated due to the high error rate of Taq DNA polymerase. The PCR conditions were the same as those described by Zhou et al. (41), and the 100-µl PCR mixture contained 50 mM KCl, 20 mM Tris-HCl (pH 8.0), 1.5 mM MgCl₂, 0.01% gelatin, 0.2 mM each deoxynucleoside triphosphate, 200 ng of each primer, 5 U of Taq DNA polymerase, and 5 ng of pXZ29 plasmid. The amplified DNA fragments were cut with PstI and NcoI and reinserted into pXZ29.

Protein purification and DNA binding. Wild-type and mutant CAP proteins were purified by cyclic AMP (cAMP) affinity chromatography as described previously (39), yielding proteins more than 95% pure as judged from sodium dodecyl sulfate-acrylamide gel electrophoresis. The DNA binding affinities of these CAP proteins were measured by the DNA migration retardation assay (38). The 250-bp DNA fragments used in the assay contain ara p_BAD, which was amplified from p10+CAP by PCR with the primers 5'-AATAGGCGTATCACGAGGCCC-3' and 5'-GATGGGGAGTAAGCTTGGATTCCAATTGCAATCGC-3'. The DNA binding buffer contained 50 mM KCl, 25 mM sodium HEPES (pH 7.4), 2.5 mM MgCl₂, 2.5 mM dithioerythritol, 100 µM cAMP, 100 µg of bovine serum albumin per ml, 0.1 mM potassium EDTA, 5% glycerol, and 20 µg of calf thymus DNA per ml. CAP was incubated with 0.03 nM labeled araBAD DNA fragments at 37°C for 10 min before the reaction products were subjected to electrophoresis.

	RESULTS

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CAP can activate the araBAD promoter without the involvement of upstream DNA. DNA upstream of the araBAD promoter participates in transcription repression by DNA looping (9). The loop is formed when AraC protein binds to the upstream araO₂ and the downstream araI₁ half-sites. CAP helps disrupt the loop to unlock the promoter (27). It is not known, however, how CAP activates transcription when the araO₂ site has been deleted. One possibility is that AraC uses a hidden upstream binding site or nonspecific DNA and still forms a DNA loop that CAP helps to break. To rule out this possibility, we used just the DNA binding domain of AraC (7). This protein fragment does not dimerize and therefore cannot form a DNA loop. The domain can, however, bind to p_BAD, with a special araI₁* half-site replacing araI₂. araI₁* binds AraC considerably more tightly than araI₂ binds AraC (32). As shown in Fig. 2, even in this case, CAP still stimulates the araBAD promoter. Apparently this stimulating activity of CAP is independent of AraC-induced DNA looping.

View larger version (29K): [in this window] [in a new window]   FIG. 2.   CAP activation at ara pBAD without the aid of upstream DNA. (Top) In the presence of wild-type AraC, CAP can activate ara pBAD-I1-I1* about 12-fold. (Middle) In the presence of the AraC DNA binding domain, CAP can also activate transcription from ara pBAD-I1-I1* about 10-fold. (Bottom) At ara pBAD-I1-I1*, with an additional CAP binding site to bend DNA away from RNA polymerase, CAP can still activate about 12-fold. The promoter activity was measured by the -galactosidase assay in SH322 cells or in SH321 cells with the AraC DNA binding domain expressed from plasmid pGBO21B. Arabinose was not present. The transcription activation by CAP was calculated by comparing two promoters with or without CAP binding sites.

CAP does not affect AraC binding in the unlooped state. CAP does not significantly activate p_BAD in the absence of AraC (data not shown). It is conceivable, then, that CAP may just stabilize AraC binding to DNA or force AraC to bend to reach a conformation from which it can activate transcription. Either way, CAP would influence the binding of AraC to DNA. To assess possible CAP-AraC interactions in vivo, we compared the amount of AraC binding in the presence and absence of CAP. We performed DMS footprinting at araBAD promoters with and without the CAP binding site. Neither promoter possessed the araO₂ half-site, and thus CAP can stimulate them only directly, not by assisting in loop breaking. If CAP affects the amount of AraC binding, we should be able to detect a difference of AraC footprinting between these two promoters.

View larger version (56K): [in this window] [in a new window]   FIG. 3.   In vivo DMS footprinting shows that AraC binding is not affected by CAP. The left three lanes show the DMS footprinting of plasmid p10, which has the ara pBAD-I1-I1* promoter without a CAP binding site. The right three lanes show the footprinting at p10+CAP plasmid, which has the araBAD-I1-I1* promoter with a CAP site. The footprinting was done in SH321 or SH322 cells.

Isolation of crp pc mutants for the araBAD promoter. Having excluded other reasonable possibilities, we considered the possibility that CAP directly interacts with RNA polymerase through protein-protein interactions. If this were the case, we should be able to isolate mutants of CAP defective in the interaction. The desired mutants should still bind DNA normally, however, and could be called pc mutants.

Characterization of the CAP pc mutants. The CAP mutants we isolated repress the p_lacUV5-O^CAP promoter as well as wild-type CAP (Table 1). This indicates that they are not defective in DNA binding, although it is possible that L150P and Q164P owe their reduced activation effects merely to weakened DNA binding. In vivo analysis shows that all these CAP mutants are defective in activating the ara p_BAD promoter, in both the presence and absence of arabinose (Table 1). These results show that the CAP mutants we isolated meet the criteria of pc mutants for p_BAD.

View larger version (31K): [in this window] [in a new window]   FIG. 4.   DNA migration retardation assay of CAP binding to DNA. The concentration of DNA fragments containing the ara pBAD-I1-I1* promoter was 0.03 nM. The concentrations of wild-type (wt) CAP protein from lanes 1 to 5 were 6.3, 12.5, 25, 50, and 100 nM. The concentrations of CAP mutant D67G from lanes 6 to 11 were 1.3, 2.5, 5, 10, 20, and 40 nM. The occupancy of CAP binding site increased with higher concentrations of CAP, and the equilibrium dissociation constant was calculated as shown in the text.

	DISCUSSION

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The role of CAP as a coactivator at the araBAD promoter has been investigated in this study. We showed that CAP can stimulate transcription at this promoter in a loop-independent way that does not involve altering AraC DNA binding. We have identified two regions of CAP that are important for activation of the araBAD promoter and the related araFGH and rhaBAD promoters. In light of extensive work by others, our results suggest that CAP directly interacts with RNA polymerase at ara p_BAD, ara p_FGH, and rha p_BAD.

At the araBAD promoter, the AraC protein binding site is centered at position 52.5 and CAP is centered at position 93.5. How the CAP protein manages to activate transcription from so far away is not clear. AraC protein represses p_BAD transcription by forming a DNA loop, and previous experiments have shown that CAP can help open the repression loop and thereby stimulate promoter activity (15, 27), but this does not account for all the CAP activation effect.

An attractive model for CAP activation at ara p_BAD is that CAP directly interacts with RNA polymerase. This view was cast in doubt, however, when it was reported that the AR1 mutation H159L did not reduce the CAP activation at ara p_BAD (37). This mutation has been shown to disrupt a large number of promoters where CAP directly interacts with RNA polymerase (10). The confounding study was done with the wild-type araBAD operon, and DNA looping was possible (37). Hence, substantial CAP activation could have been seen merely as a result of opening the DNA loop. This may have obscured any direct effects of the AR1 mutation. On the other hand, the result did not exclude the possibility that other regions on CAP were responsible for interaction with RNA polymerase at ara p_BAD.

We attempted to test the direct-interaction model by isolating CAP mutants defective in transcription activation at ara p_BAD but not defective in DNA binding. Interestingly, one group of mutants we isolated had altered residues in or near the activating region one of CAP, AR1. Many lines of evidence have suggested that at class I and II promoters, where CAP is the sole activator, AR1 of CAP interacts with the C-terminal domain of the RNA polymerase alpha subunit (5, 10). CAP uses this interaction to recruit RNA polymerase to the promoter and increase its initial binding to DNA. The same mechanism probably applies at the araBAD promoter.

If CAP contacts the alpha subunit of RNA polymerase, how does it reach past the interposed AraC protein? If the intervening protein bends the DNA sharply, such an interaction may easily take place. Previous studies have shown that at some promoters with CAP bound around position 91 and another activator at 42, AR1 was also required for CAP to activate transcription (6, 23). Since the C-terminal domain of the alpha subunit is tethered to RNA polymerase through a flexible linker (3, 20), it is conceivable that this domain can reach over another protein to contact the AR1 of the upstream CAP. At ara p_BAD, AraC-induced DNA bending may also help to move the upstream CAP closer to contact RNA polymerase.

The identification of the second group of crp pc mutants at the araBAD promoter was unexpected. The mutations clustered around Lys-52, which is contained in AR3 (5). A number of amino acid substitutions at Lys-52, including the one we isolated, can improve CAP activation at class II promoters (2). Also, a previous cross-linking study indicated that Lys-52 is positioned very close to the sigma subunit of RNA polymerase at these promoters (21). It was suggested that mutations at Lys-52 expose a nonnative activating region that can interact with the RNA polymerase sigma subunit to activate transcription at class II promoters (5). It is unclear, however, what the role of AR3 at ara p_BAD is. Our AR3 mutation K52E reduced CAP activation at p_BAD, which could mean that this mutation disrupted an existing activating region of CAP instead of revealing one. Recent studies have indicated that the two alpha subunits of RNA polymerase can each contact upstream activating elements (28). Possibly, one alpha subunit interacts with AR1 and another interacts with AR3 of CAP. It has also been proposed that at class II promoters, functional AR3 interacts with residues in the region of the sigma subunit from positions 590 to 600 (5). Previous studies identified sigma mutations at His-596, which made ara p_BAD independent of CAP (18), suggesting a sigma-AraC interaction.

A number of the mutations we isolated in the AR3 region are buried inside the protein. These buried amino acids probably cannot contact another protein directly, yet they reduce the CAP activation at p_BAD. Perhaps they induce conformational changes in CAP that disrupt the surface-exposed AR1 or AR3 or their accessibilities. We should also point out that residues in this area have been implicated in induction of CAP by cAMP (1). Recently, a second cAMP pocket was found to exist near the DNA binding domain of CAP (31). Its occupancy by cAMP or the effects of its occupancy could be affected by our mutations.

It is not surprising that the CAP AR1 mutants that are defective in transcription activation at ara p_BAD also affect two related promoters, ara p_FGH and rha p_BAD. AR1 of CAP probably directly contacts RNA polymerase at these two promoters. Mutations near AR3 only slightly disturb CAP activation at p_FGH, however, as might be expected since this is a class II CAP-dependent promoter that also contains upstream AraC binding sites, and AR3 and the surrounding region in wild-type CAP does not directly participate in transcription activation at class II promoters. At the araBAD and rhaBAD promoters, however, mutations in the AR3 region strongly reduce CAP activation. These two promoters both have another activator protein positioned between CAP and RNA polymerase, as opposed to class II CAP-dependent promoters, where CAP binds adjacent to RNA polymerase.

Our results indicate that CAP plays a direct role in transcription activation at ara p_BAD. We have demonstrated that CAP can activate ara p_BAD without altering DNA looping or AraC binding. A number of CAP pc mutants defective at p_BAD were isolated, and they fell into the regions of CAP known to interact with RNA polymerase at many promoters. These lines of evidence suggest that direct CAP-RNA polymerase interactions also occur at ara p_BAD.