On the Role of the Multiple Regulatory Elements Involved in the Activation of theEscherichia coli malEpPromoter

J. Mol. Biol. (1996) 264, 852–862

On the Role of the Multiple Regulatory Elements Involved in the Activation of the Escherichia coli malEp Promoter Evelyne Richet Unite´ de Ge´ne´tique Mole´culaire URA CNRS 1149 Institut Pasteur 25 rue du Dr. Roux 75724 Paris Cedex 15 France

Activation of malEp and malKp, two divergent promoters from Escherichia coli, depends on the synergistic action of MalT and CRP. The reaction involves a common regulatory region located in between and comprising multiple binding elements for both regulatory proteins. The binding of MalT and CRP to this region is known to result in the formation of a higher-order structure that is responsible for malKp activation. This paper presents genetic data which together with previous results, provide compelling evidence that this higher-order structure is also responsible for malEp activation. The role(s) that this structure or elements thereof play in the activation of malEp is analysed by monitoring both the occupancy of the proximal MalT sites (sites 1 and 2) and the activity of different malEp variants in strains containing increasing amounts of active MalT. A truncated malEp promoter comprising only MalT sites 1 and 2 forms a minimal MalT-dependent promoter whose activity is limited by the occupancy of these sites. One role of the higher-order structure formed by MalT and CRP when bound to the entire regulatory region is to ensure high occupation of MalT sites 1 and 2, but it is not its only function. Some elements of this structure, namely the CRP site 1, located at −76.5, and the distal MalT sites, seem to play a direct role in malEp activation besides their participation in the assembly of the higher-order structure. 7 1996 Academic Press Limited

Keywords: MalT; CRP; Escherichia coli; transcription activation; DNA loops

Introduction Prokaryotic transcriptional activators act either alone or synergistically, depending on the promoter. The transcription activation process is beginning to be well understood at the mechanistic level for the s70-dependent promoters controlled by one activator. The action of the activator protein generally involves a direct contact with RNA polymerase, and requires that its binding site be correctly positioned with respect to the promoter −35/−10 elements (Busby & Ebright, 1994; Bushman et al., 1989; Kolb et al., 1993; Mencı´a et al., 1993). In contrast, less is known about synergistic activation. So far, only a few coactivation systems have been extensively studied and the results obtained suggest two different scenarios. According to the first one, synergy seems to result from independent and direct interactions of each Abbreviations used: CRP, cAMP receptor protein; DMS, dimethyl sulphate. 0022–2836/96/500852–11 $25.00/0

activator molecule with the RNA polymerase. The location of the activator binding sites and their mode of action are then identical with what is observed for promoters at which they act alone (Joung et al., 1993, 1994; Busby et al., 1994; Scott et al., 1995). In the second scenario, one activator plays the role of the primary activator, i.e. acts directly upon the RNA polymerase, while the second activator helps the primary activator to form an active complex with promoter DNA (Lobell & Schleif, 1991; Richet et al., 1991). In this case, the binding site of the second activator is not located at a position that allows the protein to act directly upon the RNA polymerase and the mode of action of the activator differs from what is observed for promoters at which it acts alone. The analysis of new coactivation systems might reveal alternative regulatory schemes. malEp and malKp are two divergent Escherichia coli promoters which control the expression of malEFG and malK-lamB-malM, the operons encoding the components of the maltodextrin transport 7 1996 Academic Press Limited

malEp Activation

system. Their activation depends on the synergistic action of MalT, the activator of the maltose regulon (Schwartz, 1987), and CRP, a global regulator controlling the utilization of carbon sources (Kolb et al., 1993). Both promoters show some activity in the presence of MalT alone while they are completely inactive in the presence of CRP alone. Genetic and biochemical studies have shown that malEp and malKp share the same 210 bp regulatory region which is located between their transcription start sites and comprises multiple binding sites for both activators (Figure 1). The mechanism whereby MalT and CRP coactivate malKp is now well understood; it follows the second scenario mentioned above (Richet et al., 1991). The primary activator is MalT which activates malKp when bound to the 3'/4'/5' set of sites present in the promoter proximal region. However, occupation of this series of sites is prevented by MalT binding to the overlapping 3/4/5 set of sites for which MalT has a higher intrinsic affinity, and whose occupation does not lead to promoter activation because it is incorrectly located with respect to the promoter −35/−10 elements. Occupation of the productive set of MalT sites (3'/4'/5 ') instead of the competing 3/4/5 set of sites strictly depends on the formation of a higher-order structure wherein MalT is thought to bind cooperatively sites 3'/4'/5' and the distal sites 1 and 2. Formation of this structure is disfavored when MalT is present alone and the role of CRP in malKp activation is to facilitate the repositioning of MalT onto sites 3'/4'/5' by stabilizing this higher-order structure. As shown by bend-swap experiments, CRP mainly acts by

853 bending DNA in the central region (Richet & So gaard-Andersen, 1994). Negative DNA supercoiling also plays a critical role in the assembly of this higher-order structure (Richet & Raibaud, 1991). In contrast, the mechanism whereby MalT and CRP coactivate malEp remains elusive. The structure of malEp does not resemble that of any other known MalT-dependent promoters (VidalIngigliardi et al., 1991). The only characteristic that malEp shares with the other MalT-dependent promoters is the presence of a MalT site centred at −37.5 or −38.5 and similarly orientated. This site is thought to provide an anchorage point for the RNA polymerase (Danot & Raibaud, 1994; Danot et al., 1996). The inability of CRP to activate malEp on its own is most likely due to the fact that none of the CRP sites is located at a permissive position (Figure 1; Gaston et al., 1990; Ushida & Aiba, 1990; Valentin-Hansen et al., 1991). Several pieces of evidence suggest that malEp activation might involve the same nucleoprotein complex as malKp activation. First, deletion analyses showed that the two promoters share the same regulatory region (Raibaud et al., 1989). Second, they exhibit the same sigmoidal responses to increasing concentration of MalT in vitro (Richet & Raibaud, 1991). Third, mutations inactivating MalT sites 1 and 2 and the three CRP sites affect the activity of both promoters in parallel (Vidal-Ingigliardi & Raibaud, 1991; Vidal-Ingigliardi et al., 1991). However, evidence that the two promoters depend on exactly the same regulatory elements is still lacking: it is not yet clear which of the distal set of MalT sites is implicated in

Figure 1. Structure of the malEp-malKp region. The malEp-malKp region is drawn to scale with the −10 and −35 promoter elements (hatched boxes), the MalT binding-sites (filled, arrowed boxes) and the CRP binding-sites (the open boxes) indicated. The transcription start sites (arrow heads) of malEp and malKp are 271 bp apart. The base-pairs are numbered with respect to the malEp transcription start site. For the sequence of the malEp-malKp region, see Raibaud et al. (1989). The 3/4/5 series of sites is staggered by 3 bp from the 3'/4'/5' series of sites. For their exact location, see Richet et al. (1991). The end points of the malEp upstream deletions malEpD64 and malEpD92 (Raibaud et al., 1989) are indicated.

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malEp activation. Here, we present genetic data indicating that malEp primarily depends on sites 3', 4' and 5', thereby providing further evidence that activation of both promoters relies on the same higher-order structure. An in vivo analysis of the role(s) this complex or elements of this complex might play in malEp activation is also reported.

Results malEp activation depends primarily on sites 3', 4' and 5' Each set of three MalT sites (3/4/5 and 3'/4'/5') forms a functional unit to which MalT binds cooperatively (Danot & Raibaud, 1994; Richet et al., 1991). As a result, the specific inactivation of one site within a given unit is generally sufficient to reduce MalT binding to this unit without precluding MalT binding to the overlapping set of MalT sites (Richet et al., 1991). The role of the upstream sets of MalT sites in malEp activation was assessed by examining in vivo the effect of mutations specifically inactivating either set of sites on promoter activity. For this purpose, I used pOM82, a low copy number plasmid carrying a transcriptional fusion between malEp+ and the lac operon and I measured the amount of b-galactosidase made by a Dlac strain (pop2226) bearing pOM82 or derivatives thereof. The cells were grown in minimal medium in the presence of glycerol and maltose, i.e. under conditions in which both MalT and CRP are active. The results obtained with the different mutants were compared to the residual level of activity observed with a promoter lacking both sets of distal MalT sites. malEpD92 (see Figure 1) could be used as a reference because the residual malEp activity observed when the distal MalT sites are deleted is the same whether or not the CRP sites 2 and 3 are still present (Raibaud et al., 1989). Figure 2(a) shows that mutations in sites 4 or 5 increase malEp activity slightly while mutations in sites 3', 4' or 5' reduce promoter activity to 65 to 80% of the wild-type level. Since inactivation of one site does not completely inactivate the corresponding set (Richet et al., 1991), promoter mutants in which two sites of a given set had been specifically inactivated by point mutations were also assayed. The results obtained were similar to those obtained with single-site mutants (Figure 2(a)). A variant in which both sites 3 and 4 had been inactivated was still slightly more active than the wild-type while a variant in which both sites 3' and 4' had been destroyed and which can be considered as harbouring only the 3/4/5 set of sites (Richet et al., 1991), still displayed a high level of activity compared to malEpD92 (50% versus 16%). In addition, a variant in which both functional units (3/4/5 and 3'/4'/5') had been inactivated by the destruction of sites 3, 4, 3' and 4' displays the same level of activity as malEpD92 (Figure 2(a)), thereby excluding the possibility that the malEp stimulation still observed when either set of distal MalT sites is

Figure 2. Effect of mutations in the distal MalT sites on the in vivo activity of malEp. The amount of b-galactosidase made by strain pop2226 harbouring various derivatives of pOM82 and grown in the presence of glycerol or glycerol + maltose were determined as described in Materials and Methods. The results are expressed relative to the value obtained for induced wild-type malEp (16,000 units). The point mutations malEpKp30, -42, -50, -300, -400, -500 are described by Richet et al. (1991). The effect of different mutations in the distal MalT sites were examined (a) in the malEp+ context and (b) in the malEpKp1,2,3 context. Crosses symbolize the malEpKp1, -2 and -3 mutations, which correspond to 3 bp substitutions and which inactivate the CRP sites 1, 2 and 3, respectively (Vidal-Ingigliardi & Raibaud, 1991).

malEp Activation

inactivated is due to some other sequence element and not to the remaining set. Altogether, these results therefore indicate that the stimulatory effect of the distal region on malEp implicates the same functional units (3/4/5 and 3'/4'/5) as for malKp regulation, and that either set of sites can stimulate malEp although 3'/4'/5' is clearly more effective than 3/4/5. The relative ability of the two sets of MalT sites to activate malEp was also examined under conditions in which the concentration of active MalT was limiting, i.e. in the presence of glycerol as the sole carbon source (Vidal-Ingigliardi & Raibaud, 1991). Under these conditions, all but one (malEpKp500) of the single or double mutants of the 3'/4'/5' set of sites scarcely showed any activity while single or double mutants of the 3/4/5 set of sites were as active as malEp+ (Figure 2(a)). The variant carrying only the 3'/4'/5' series of sites (mutant malEpKp30,42) was five times more active than that carrying only the 3/4/5 series of sites (mutant malEpKp300,400). A variant in which both sets of distal MalT sites had been inactivated (mutant malEpKp30,42,300,400) did not show any significant activity (Figure 2(a)). Hence, when MalT is limiting, malEp activation critically depends on the 3'/4'/5' set of sites. The simplest interpretation of these observations is that the oligomeric assembly resulting from the cooperative binding of MalT onto sites 1/2 and sites 3'/4'/5' can also form when MalT occupies sites 1/2 and 3/4/5, albeit less easily, and that both types of complex can activate malEp (see Discussion). The observation that the malEpKp500 mutation has a weaker deleterious effect on malEp activity than mutations malEpKp300 or -400 (Figure 2(a)) is most likely due to the fact that specific inactivation of MalT site 5' might not be sufficient to preclude any cooperative binding of MalT onto sites 1/2 and 3'/4'. The coupling between occupation of sites 3'/4' and occupation of site 5' indeed seems to be somewhat loose: simultaneous occupation of sites 3'/4' and 5 has been observed in vitro; moreover, cooperative binding of MalT onto sites 1/2 and 3'/4' has been observed in vitro in the absence of site 5' (Richet et al., 1991). To determine whether the higher efficiency of the 3'/4'/5' set of sites in promoting assembly of a higher-order structure depended on the presence of CRP, I measured the effect of the distal sets of MalT sites on malEp in the absence of functional CRP sites. For this purpose, I constructed malEp variants containing point mutations destroying the three CRP sites (malEpKp1, -2, and -3) and mutations in either sites 3 and 4, sites 3' and 4', or sites 3, 4, 3' and 4', and assayed them in vivo in the presence of glycerol and maltose. Figure 2(b) shows that the 3'/4'/5' set of sites still had a stronger stimulatory effect on malEp than the 3/4/5 set. Moreover, inactivation of both distal sets of MalT sites together with the three CRP sites abolishes promoter activity as efficiently as deletion malEpD64 (Figure 2(b)), thereby indicating that, in

855 the absence of functional CRP sites, malEp activity is fully dependent on the presence of either set of distal MalT sites. Occupation of MalT sites 1 and 2 requires the presence of upstream regulatory elements As mentioned above, the truncated malEpD64 promoter, which comprises only MalT sites 1 and 2, is inactive. In contrast, a synthetic promoter containing the malPp −35/−10 region and two consensus MalT sites located at the same position and in the same orientation as the MalT sites 1 and 2 present in malEp displays a significant level of MalT-dependent activity (Danot & Raibaud, 1993). Hence, this suggests that malEpD64 might actually represent a minimal MalT-dependent promoter that is inactive because of a too low occupation of MalT sites 1 and 2, and that the role of the upstream elements in malEp activation might simply be to ensure high occupation of MalT sites 1 and 2 through the assembly of a higher-order structure with MalT and CRP. To assess the occupancy of MalT sites 1 and 2 in malEpD64, I performed in vivo dimethyl sulphate (DMS) protection experiments on isogenic DmalT and malT + strains harbouring pOM82 or pOM82malEpD64 and grown in the presence of glycerol and maltose as for promoter assays. After plasmid isolation and strand cleavage at the methylated positions, the methylation pattern of the bottom strand of the malEp-proximal region was revealed by using the primer-extension technique (Sasse-Dwight & Gralla, 1991). Comparison of the patterns obtained for malEp+ shows that MalT sites 1 and 2 of malEp+ are occupied by MalT in a malT + strain (Figure 3(a)). The most characteristic features indicating occupation of these sites are the inversion of the methylation ratio of the GG doublet at −37 and −38, and the protection of the G at −51. The in vivo DMS protection pattern of MalT sites 1 and 2 in malEp+ is qualitatively similar to that obtained in vitro (Richet & Raibaud, 1991), although it is weaker than that observed in vitro under saturating conditions, which suggests that the MalT sites 1 and 2 of malEp+ are only partially occupied in vivo, even under conditions of full induction. In contrast, the footprint obtained with malEpD64 in a malT + strain is indistinguishable from that obtained with malEpD64 or malEp+ in a DmalT strain. Thus, occupation of sites 1 and 2 is much lower in malEpD64 than in malEp+. To ensure that the occupation of MalT sites 1 and 2 observed with malEp+ was not due to a stabilizing effect of RNA polymerase bound at malEp and/or malKp, I verified that MalT sites 1 and 2 were similarly occupied in the absence of functional −10 sequences. As shown in Figure 3(b), the disruption of the −10 sequences of both malEp and malKp by mutations malEpKp60 and -70 (see Figure 1), which abolishes the activity of both promoters (data not shown), does not change the degree of protection of MalT sites 1 and 2. Altogether, these data

856

Figure 3. In vivo occupation of MalT sites 1 and 2 in malEp+ and malEpD64. In vivo DMS protection experiments were performed by using isogenic DmalT and malT + strains, pop2150 and pop2226, respectively, bearing pOM82 or derivatives of it, as indicated on the Figure. The methylation pattern shown is that of the bottom strand. The residues are numbered with respect to the malEp transcription start site. The arrowed boxes indicate the position of MalT sites 1 and 2. The open and filled circles mark the purine residues whose methylation by DMS is diminished and increased, respectively. The asterisk marks a G methylation enhancement (at − 26) which is observed with a malEp+ variant when present in a malTp+ strain, and which most likely results from RNA polymerase binding.

demonstrate that occupation of MalT sites 1 and 2 depends on the presence of upstream regulatory elements. Full occupation of MalT sites 1 and 2 partially reactivates malEp D64 To challenge the simple hypothesis that the upstream regulatory elements only serve to ensure high occupation of the MalT sites 1 and 2, I determined whether occupation of MalT sites 1 and 2 could reactivate malEpD64 to wild-type level. Complete occupation of the MalT sites 1 and 2 of malEpD64 was achieved by using strains harbouring the malTp1 malTp7 mutations, which cause MalT overproduction. malTp1 and malTp7 are mutations of the malT gene which together increase

malEp Activation

Figure 4. Occupation of MalT sites 1 and 2 in malEpD64 in strains containing increasing amounts of active MalT protein. In vivo DMS protection experiments were performed on isogenic strains containing pOM82 or derivatives of it, as described in Material and Methods. The methylation pattern shown is that of the bottom strand. Lane 1, pop2226 (pOM82); lane 2, pop2150 (pOM82malEpD64); lane 3, pop2226 (pOM82malEpD64); lane 4, pop4133 (pOM82malEpD64); lane 5, pop3971 (pOM82malEpD64).

its expression 30 times (Chapon, 1982). (Note that such an increase does not necessarily result in an equivalent increase in the concentration of active MalT). The strains used are either malTp1 malTp7 malPQ− or malTp1 malTp7 malPQ+, and both are isogenic to pop2226, the malTp+ malPQ− strain used in the above experiments and hereafter called the reference strain. The presence of a wild-type malPQ+ operon, which encodes enzymes involved in the catabolism of maltodextrins, further increases the concentration of active MalT in a malTp1 malTp7 background (see below), possibly through an effect on the production of maltotriose (the true inducer) from maltose. In vivo DMS protection analyses showed that in the malPQ− overproducing strain, the MalT sites 1 and 2 of malEpD64 were protected to the same extent as the MalT sites 1 and 2 of malEp+ in the reference strain, whereas their protection was complete in the malPQ+ overproducing strain (Figure 4). Promoter assays performed in parallel revealed that forced occupancy of MalT sites 1 and 2 does reactivate malEpD64 but only to a limited extent (Table 1). malEpD64 was most active in the malTp1 malTp7 malPQ+ strain in which protection of the

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Table 1. Acivity of malEp variants in strains overproducing MalT malEp variant

Strains +

malEp+ malEpD64 malEpD92 malEpKp1,2,3,I3

malTp malPQ− 100 E0.5 16 29

malTp1 malTp7 malPQ− 123 5 71 42

malTp1 malTp7 malPQ+ 93 8 55 39

The level of b-galactosidase activity in isogenic malTp+ malPQ− (pop2226), malTp1 malTp7 malPQ− (pop4133), and malTp1 malTp7 malPQ+ (pop3971) strains harbouring variants of pOM82 and grown in the presence of glycerol and maltose was determined as described in Materials and Methods. The results are expressed relative to the value obtained for induced wild-type malEp (16,000 units).

MalT sites 1 and 2 was complete. However, its activity (8%) was still low compared to that of malEp+. Controls showed that malEp+ is slightly more active in the malPQ− overproducing strain while it is marginally less active in the malPQ+ overproducing strain (Table 1), which indicates that MalT overproduction does not lead to promoter inhibition, at least in the case of the wild-type promoter. From these experiments, it can be concluded that malEpD64 actually corresponds to a minimal MalT-dependent promoter whose activity is not just limited by the weak intrinsic affinity of MalT for sites 1 and 2. The fact that complete occupation of MalT sites 1 and 2 only partially reactivates malEpD64 indeed strongly suggests that some of the upstream elements play a specific role in malEp activation. The distal MalT sites play an additional role in malEp activation The possibility that the MalT molecules bound to either set of distal sites might play a direct role in malEp activation was tested by examining whether, when achieved in the presence of the distal MalT sites, full occupation of the proximal MalT sites would restore promoter activity to a higher level than that observed with malEpD64. The promoter used was the malEpKp1,2,3,I3 variant, in which the three CRP sites had been inactivated by point mutations (malEpKp1, -2, and -3) and which also contained a 3 bp insertion (malEpKpI3) in the central region to bring the higher-affinity set of MalT sites (3/4/5) in register with MalT sites 1 and 2 (Recall that the two overlapping sets of MalT sites are staggered by 3 bp). This variant is fivefold more active than the malEpKp1,2,3 variant and is 29% as active as malEp+ (Figure 2(b) and Table 1). Footprinting analyses revealed that, in pop2226, the reference strain, the MalT sites 1 and 2 of the malEpKp1,2,3,I3 variant were occupied to the same degree as those of malEp+ and that they were fully occupied in the MalT overproducing strains (Figure 5(a)). The occupancy of MalT sites 1 and 2 does not result from stabilization by RNA polymerase bound to malEp because the inactivation of the −10 element of malEp does not alter the degree of protection of MalT sites 1 and 2 of the

malEpKp1,2,3,I3 variant in pop2226 (Figure 5(a)). Promoter assays showed that MalT overproduction increases the activity of the malEp1,2,3,I3 variant only slightly (42 to 39% versus 29%; Table 1). The fact that malEpKp1,2,3,I3 was not fully active in the MalT overproducing strains was not due to the presence of an excess of MalT because intermediate values were obtained when the variant was assayed in strains overproducing MalT to a lesser extent (in malTp1 malPQ− or malTp7 malPQ− strains; data not shown). Under conditions of full occupancy of MalT sites 1 and 2, the malEpKp1,2,3,I3 variant is thus fivefold more active than malEpD64 (compare the value obtained with the malEpKp1,2,3,I3 mutant when present in the malPQ− overproducing strain (42%) to that obtained with malEpD64 when present in the malPQ+ overproducing strain (8%)). This indicates that the distal set of MalT sites plays an additional role in malEp activation besides aiding MalT to bind to sites 1 and 2. However, the fact that in pop2226, the malEpKp1,2,3,I3 variant is only 29% as active as the wild-type promoter, while displaying similar occupation of MalT sites 1 and 2, suggests that complete occupation of MalT sites 1 and 2 in the presence of the distal MalT sites is not sufficient to restore malEp activity fully and that one of the CRP sites also plays a direct role in promoter activation. CRP bound to site 1 plays a specific role in malEp+ activation The observation that CRP site 1 still strongly stimulated malEp in the absence of any distal MalT sites (Raibaud et al., 1989; Vidal-Ingigliardi et al., 1991) suggested that the CRP molecule bound to site 1 might play an active role in malEp activation, besides its structural role in the assembly of the active complex. A direct role for CRP sites 2 or 3 was considered unlikely given that, when the distal MalT sites have been deleted, the residual malEp activity remains unchanged if the deletion also removes CRP sites 3 or 2 + 3 (Raibaud et al., 1989). The hypothesis of a direct role for CRP site 1 was tested by examining whether occupation of MalT sites 1 and 2 in the presence of CRP site 1 increased malEp activity to a higher level than that observed with malEpD64. These experiments were performed

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for malEp+ in the reference strain (Figure 5(b)). The fact that in the malPQ− overproducing strain, malEpD92 was almost as active as malEp+ in pop2226 for a similar occupancy of MalT sites 1 and 2, indicates that filling MalT sites 1 and 2 in the presence of CRP site 1 efficiently restores promoter activity. Interestingly, malEpD64 and malEpD92 displayed similar partial occupancy of MalT sites 1 and 2 when present in the malPQ− overproducing strain, while malEpD92 was 14-fold more active than malEpD64. This indicates that in the malEpD92 context, CRP does not cause increased transcription by stabilizing MalT at sites 1 and 2.

Discussion A unique nucleoprotein assembly activates malEp and malKp

Figure 5. Occupation of MalT sites 1 and 2 in the malEpD92 and malEpKp1,2,3,I3 variants in strains containing increasing amounts of active MalT protein. In vivo DMS protection experiments were performed on isogenic strains containing pOM82 or derivatives of it. The methylation pattern shown is that of the bottom strand. Lane 1, pop2226 (pOM82); lane 2, pop2150 (pOM82malEpKp1,2,3,I3); lane 3, pop2226 (pOM82malEpKp1,2,3,I3); lane 4, pop2226 (pOM82malEp Kp60,1,2,3,I3,70); lane 5, pop4133 (pOM82malEp Kp1,2,3,I3); lane 6, pop3971 (pOM82malEpKp1,2,3,I3); lane 7, pop2226 (pOM82); lane 8, pop2150 (pOM82 malEpD92); lane 9, pop2226 (pOM82malEpD92); lane 10, pop4133 (pOM82malEpD92); lane 11, pop4133 (pOM82 malEpD64).

with the truncated malEpD92 promoter variant (see Figure 1). As shown in Table 1, MalT overproduction strongly enhances the activity of malEpD92. The variant was most active in the malPQ− overproducing strain in which it was 71% as active as malEp+ in pop2226. A further increase in the intracellular concentration of active MalT protein appeared to be slightly inhibitory (Table 1). When assayed in a malTp1 malPQ− strain, which moderately overproduces MalT, malEpD92 was twofold less active than in the malTp1 malTp7 malPQ− strain (data not shown). Footprinting analyses showed that the MalT sites 1 and 2 of malEpD92 are essentially unoccupied in pop2226 while in the malPQ− overproducing strain they were protected to the same extent as observed

The MalT sites 1 and 2, as well as the CRP sites 1, 2 and 3 had all been shown to play a role in malEp activation (Vidal-Ingigliardi & Raibaud, 1991; Vidal-Ingigliardi et al., 1991), but so far it was not clear which of the MalT sites present in the distal region are involved in promoter activation. By using an in vivo genetic approach, I have shown here that the distal MalT sites also function as two alternative binding units of three sites with respect to malEp activation, as observed for malKp, and that malEp primarily depends on the 3'/4'/5' set of sites. Indeed, the 3'/4'/5' set of sites is clearly the most efficient of the two sets of MalT sites in stimulating malEp at a saturating concentration of MalT, while only the sites 3'/4'/5' have the capacity to stimulate malEp when the MalT concentration becomes limiting. Hence, malEp activation primarily relies on MalT sites 1/2, 3'/4'/5' and on the CRP sites 1/2/3, i.e. on the same set of regulatory elements as malKp activation. From this, we infer that the higher-order structure responsible for malKp activation is also responsible for malEp activation. This inference is further supported by the observation that, in vitro, malEp and malKp exhibit superimposable sigmoidal responses to increasing concentration of MalT (Richet & Raibaud, 1991), which indicates that the same event of cooperative binding underlies the two activation processes. Moreover, mutations in the CRP bending loci affects malEp and malKp in parallel (Vidal-Ingigliardi & Raibaud, 1991), thereby indicating that the nucleoprotein complexes involved in the activation of each promoter have the same architecture. Altogether, these observations make it unlikely that the same ensemble of binding elements commands the assembly of two alternative higher-order structures, one mediating malEp activation, the other mediating malKp activation, and provide compelling evidence that the same nucleoprotein assembly is responsible for the activation of both promoters.

malEp Activation

The sensitivity of higher-order structure formation to the phasing between the proximal and distal sets of MalT sites is essentially determined by MalT Previous studies lead us to propose that formation of the higher-order structure at malEpmalKp is sensitive to the phasing between MalT sites 1/2 and the distal set of sites, and only occurs when MalT occupies sites 3'/4'/5' (Richet et al., 1991). However, the finding that the 3/4/5 set of sites stimulates malEp to some extent provided that the concentration of active MalT protein is high enough (i.e. in an induced malT + strain) indicates that the oligomeric structure built by MalT when occupying sites 1/2 and 3'/4'/5' can also form when MalT occupies sites 1/2 and 3/4/5, albeit less efficiently. Assembly of a looped structure based on sites 1/2 and 3/4/5 is best explained by supposing that the higher affinity that MalT displays for sites 3/4/5 compared to sites 3'/4'/5' partially compensates for the incorrect phasing between 1/2 and 3/4/5. Hence, looped structures of the 1/2-3'/4'/5' type most likely coexist with looped structures of the 1/2-3/4/5 type in vivo under inducing conditions. While the former will activate both malEp and malKp, the latter will only activate malEp. So far, it was unclear why the higher-order structure preferentially assembles when MalT is bound to sites 1/2 and 3'/4'/5'. Is the sensitivity to the relative angular orientation of the proximal and distal sets of MalT sites a property intrinsic to MalT or is it due to the increased torsional stiffness of the intervening DNA resulting from CRP binding? The observations that the 3'/4'/5' set of sites has a stronger stimulatory effect on malEp than the 3/4/5 sites regardless of the presence of functional CRP sites and that CRP similarly enhances the stimulatory effect of both sets of MalT sites, indicates that the sensitivity to the phasing is actually essentially determined by MalT. However, because the comparative assays reported here were performed in the presence of a saturating concentration of MalT, a differential effect of CRP might have been overlooked, and the possibility remains that CRP may also increase the bias toward the 1/2-3'/4'/5' looped structures via an increase in the torsional rigidity of the DNA in the central region. Role of the regulatory elements involved in malEp activation Promoter activity assays combined with analysis of the occupancy of MalT sites 1 and 2 of different malEp variants in strains producing increasing amounts of active MalT protein proved to be a successful approach to define the role of the multiple regulatory elements involved in malEp activation. This study indeed revealed the following: (1) occupation of MalT sites 1 and 2 is sufficient to promote malEp activation; (2) in vivo, under conditions of full induction, the activity of the truncated malEpD64 promoter is severely limited by

859 the degree of occupancy of MalT sites 1 and 2; (3) one role of the upstream regulatory elements is to stabilize MalT at sites 1 and 2; (4) full occupation of these sites is not sufficient, however, to ensure full promoter activity; (5) CRP site 1 and the upstream MalT sites can independently increase the capacity of MalT bound to sites 1 and 2 to stimulate malEp; and (6) this effect of CRP bound to site 1 does not involve stabilization of MalT at sites 1 and 2. That malEpD64 represents a minimal MalT-dependent promoter displaying some activity provided that the MalT concentration is high enough was not unexpected since Danot & Raibaud (1993) had shown that malPp701, a synthetic promoter containing two MalT sites placed as in malEpD64 exhibits significant MalT-dependent activity. Such a construct represents one of the three types of MalT-dependent promoters with respect to the number, location and orientation of the MalT binding sites. Together with the fact that MalT sites 1 and 2 are the regulatory elements whose inactivation has the most severe effect on malEp (mutation in MalT sites 1 or 2 reduce malEp activity to less than 5% of the wild-type level (VidalIngigliardi et al., 1991), the observation that their occupation is sufficient to trigger initiation of transcription indicates that they play a key role in malEp activation. As mentioned in the Introduction, the MalT protomer bound to the most proximal MalT site is thought to contact RNA polymerase (Danot et al., 1996). The function of site 2 remains so far unclear. The finding that MalT sites 1 and 2 are virtually unoccupied in the absence of the upstream binding elements while they are half-protected in the malEp+ context indicates that one function of the upstream regulatory elements is to ensure high occupation of MalT sites 1 and 2. This stabilizing effect most likely results from the assembly of the higher-order structure formed by MalT and CRP at malEp-malKp. First, in vitro, MalT repositioning onto sites 3'/4'/5', i.e., formation of a higher-order structure based on sites 1/2 and 3'/4'/5', is correlated with an increase in the affinity of MalT for sites 1 and 2 (E.R., unpublished results). Second, the in vivo footprinting experiments performed with malEpD64 and malEpKp1,2,3,I3 clearly show that formation of a higher-order structure can lead to a significant stabilization of MalT onto sites 1 and 2. Finally, the possibility that the enhanced occupation of MalT sites 1 and 2 observed in the malEp+ context results from a local effect, e.g., from a direct interaction with CRP bound to the adjacent CRP 1 site, rather than from the formation of the higher-order structure is excluded by the observation that in vivo (this work) and in vitro (unpublished results) CRP binding to site 1 does not significantly increase the affinity of MalT for sites 1 and 2 in malEpD92. However, stabilization of MalT onto sites 1/2 is not the only raison d’ex tre of the higher-order structure with respect to malEp. The data presented here clearly establish that occupation of MalT sites

860 1 and 2 is indeed not sufficient to fully activate malEp, and that both the CRP site 1 and the distal MalT sites further stimulate malEp, 14 and fivefold, respectively, when the proximal MalT sites are similarly occupied. This suggests that these upstream elements play additional roles in malEp activation besides their participation to the building of the higher-order structure. What are these functions? One possibility is that the CRP molecule bound to site 1 or/and one of the MalT protomers distally bound facilitate the initiation process, either by directly contacting the polymerase or by aiding one of the MalT protomers bound downstream to interact with the enzyme. Alternatively, MalT binding to an isolated repeat (sites 1 + 2) may nucleate formation of unproductive MalT aggregates which cannot interact with RNA polymerase, and occupation of CRP site 1 and/or assembly of an oligomeric structure based on MalT sites 1/2 and 3'/4'/5' may help MalT to form a productive nucleoprotein complex at MalT sites 1/2. DNase I protection experiments have shown that MalT binding to sites 1 and 2 can lead to MalT aggregation in situ (Raibaud et al., 1989). However, it is worth noting that, were this hypothesis correct, formation of unproductive aggregates might be consequential to the artificially high concentrations of active MalT needed to achieve saturation of malEpD64; hence some of the stimulatory effects observed under these conditions might not exist under normal conditions. With regard to the specific role played by CRP bound to site 1, it is unlikely that the protein acts by preventing an aggregation phenomenon since its stimulatory effect does not increase with the concentration of MalT. Hence, I favour the idea that this effect of CRP involves a direct contact either with the MalT molecule bound to site 2 or with RNA polymerase. The absence of any cooperativity in the binding of MalT and CRP to sites 1/2 and 1, respectively, as revealed by the in vivo footprinting experiments makes it unlikely that contact with MalT bound to site 2 mediates this stimulatory effect of CRP, and lends support to the hypothesis of an interaction with the polymerase. However, the position of the CRP site 1, which is centred on position −76.5, is not expected to allow direct contacts with the polymerase: when acting alone, CRP indeed activates initiation of transcription by contacting the polymerase, but its ability to activate requires its binding site be located on the same face of the DNA helix as the polymerase binding site, i.e. at positions −41.5, −61.5, −71.5, −82.5 or −92.521 bp (Gaston et al., 1990; Ushida & Aiba, 1990; Valentin-Hansen et al., 1991). One possibility is that the MalT molecules bound to sites 1/2 locally alters the pitch of the DNA double helix, thereby bringing CRP site 1 on the same face of the double helix as the RNA polymerase binding site. One could also imagine that the CRP molecule bound to site 1 contacts the polymerase even though its binding site is not at a permissive location (Choy et al., 1995), and that the MalT protomers bound

malEp Activation

downstream transform this contact into a productive one. It is worth noting that, like malEp, the E. coli aeg-46.5 promoter, which is synergistically controlled by NarP and FNR (a CRP-related activator) has a FNR-binding site at a non-permissive position that is separated from the RNA polymerase recognition sequence by two binding sites for a second transcription factor (Darwin & Stewart, 1995; Wing et al., 1995). It will be interesting to see whether a common mechanism. underlies both synergistic phenomena. To conclude, it is important to stress the complexity of the mechanism by which MalT and CRP coactivate malEp. In this process, MalT most likely plays the role of a primary activator while CRP plays a dual role. One of its function is architectural: via binding to the three CRP binding sites and bending the DNA, it facilitates cooperative binding of MalT onto sites 1/2 and the distal sites (Richet & So gaard-Andersen, 1994), thereby ensuring high occupancy of the critical MalT sites 1 and 2. In addition, the CRP molecule bound to site 1 seems to play a direct but so far undefined role in the activation process. It will be interesting to see to what extent the latter is related to CRP function in the activation of other promoters.

Materials and Methods Strains and plasmids The E. coli strains used are listed in Table 2. Plasmid pOM82 is a derivative of pJEL126 (Valentin-Hansen et al., 1986) which contains the 478-bp EcoRI-EcoRI malEpmalKp fragment from pOM18 (Raibaud et al., 1989) inserted into the EcoRI site of pJEL126, with malEp fused to the promoter-less lacZYA operon present on the vector. Variants of pOM82 carrying mutations in the malEpmalKp region were constructed by oligonucleotide-directed mutagenesis of an M13mp7 derivative containing the same EcoRI-EcoRI malEp-malKp fragment, as described by Kunkel et al. (1987), followed by the cloning of the mutated fragment in pJEL126. In each case, the mutated fragment was entirely sequenced to verify that it did not contain other alterations.

In vivo assay of b-galactosidase The strains harbouring pOM82 or variants thereof were grown in M63 minimal medium (Miller, 1972) supplemented with 1 mg/ml thiamine, 30 mg/ml ampicillin, 0.5% glycerol and, when indicated, 0.4% maltose. The cultures were inoculated to a low-cell density (A600 1 10−4 to 9 × 10−4 ) with precultures grown at 30°C to late exponential phase in rich medium. The cultures were grown overnight at 30°C up to an A600 of 0.6 to 0.9, and b-galactosidase was assayed at 28°C as described by Miller (1972), using chloroform and 0.002% (w/v) SDS to disrupt the bacteria. The specific activity of the enzyme is given in Miller units. All values represent the average of assays performed in duplicate on at least two independent cultures and are corrected for background levels: 293 units for the pOM82 derivatives containing the malEpD64 truncated promoter, and 324 units in the other cases, as determined by measuring the amount of b-galactosidase made by strains pop2150

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Table 2. E. coli K12 strains Strain

Genotype

Source

pop2150 pop2226

araD139 D(argF-lac)U169 rpsL150 relA1 flbB5301 deoC1 ptsF25 rbsR MC4100 DmalA510 MC4100 malPp201

pop3971 pop4133

MC4100 malTp1 malTp7 MC4100 malTp1 malTp7 malPp201

MC4100

Casadaban (1976) Raibaud et al. (1983) O. Raibaud, unpublished results Chapon (1982) This work

The DmalA510 deletion is an 11 kb deletion of the malA intergenic region which removes the beginning of the malT and malP genes (Raibaud et al., 1983). The malPp201 mutation, which corresponds to the insertion of the linker 5'-GGAATTCC-3' into the HincII site present at position −26 in the malPp promoter, inactivates the promoter of the malPQ operon (O. Raibaud, unpublished results). pop4133 was constructed in two steps: (1) the malPp201 mutation was transferred from the M13malP3malPp201 phage to the chromosome of strain pop3971 (F+ ) as described by Danot & Raibaud (1994), and (2) an F− derivative of the resulting strain was obtained by selecting for M13 resistant clones.

(pOM82malEpD64) and pop2150 (pOM82), respectively when grown in the presence of maltose. The variations observed between two independent experiments did not exceed 20%.

In vivo DMS protection experiments Twelve-ml cultures, grown as described above, were treated with 3.3 ml of pure DMS, and further incubated for five minutes before a quick chilling. The cells were pelleted and plasmid DNA was extracted by using the method of Holmes & Quigley (1981). The lysate (10.9 ml) was subsequently treated with 0.2 mg/ml RNase A and 0.2 mg/ml proteinase K before precipitation with isopropanol, and proteins were removed from the DNA as described by Sasse-Dwight & Gralla (1991). Cleavage of the methylated purine residues, DNA purification and primer extension by the Klenow fragment of DNA polymerase I were performed as described by Richet & Raibaud (1991). The 5' end-labelled primer used was the EK1 oligonucleotide (Richet & Raibaud, 1991), whose sequence corresponds to the sequence extending from position +38 to position +22 on the top strand of the malEp promoter. Primer-extension analysis was generally carried out on one fifth of the material recovered from a 12 ml culture. The extension products were analysed by electrophoresis through an 8% (w/v) polyacrylamide sequencing gel. The gel was fixed, dried and subjected to autoradiography on a Kodak XAR-5 film with an intensifying screen at −70°C.

Acknowledgements I am obliged to O. Danot for stimulating discussions and his critical reading of the manuscript. I also thank A. Kolb and T. Pugsley for their comments on the manuscript and the reviewer who pointed out the analogy between malEp and the aeg-46.5 promoter.

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Edited by M. Yaniv (Received 22 April 1996; received in revised form 26 September 1996; accepted 1 October 1996)