Structural requirements for marbox function in ... - Wiley Online Library

Molecular Microbiology (1999) 34(3), 431±441

Structural requirements for marbox function in transcriptional activation of mar/sox/rob regulon promoters in Escherichia coli: sequence, orientation and spatial relationship to the core promoter Robert G. Martin,* William K. Gillette, Sangkhee Rhee and Judah L. Rosner Laboratory of Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, Bldg. 5, Rm. 333, NIH, Bethesda, MD 20892-0560, USA Summary The promoters of the mar/sox/rob regulon of Escherichia coli contain a binding site (marbox) for the homologous transcriptional activators MarA, SoxS and Rob. In spite of data from footprinting studies, the marbox has not been precisely de®ned because of its degeneracy and asymmetry and seemingly variable location with respect to the 10 and 35 hexamers for RNA polymerase (RNP) binding. Here, we use DNA retardation studies and hybrid promoters to identify optimally binding 20 bp minimal marboxes from a number of promoters. This has yielded a more de®ned marbox consensus sequence (AYnGCACnnWnnRYYAAAYn) and has led to the demonstration that some marboxes are inverted relative to others. Using transcriptional fusions to lacZ, we have found that only one marbox orientation is functional at a given location. Moreover, the functional orientation is determined by marbox location: marboxes that are 15 or more basepairs upstream of the 35 hexamer are oriented opposite those closer to the 35 hexamer. Marbox orientation and the spacing between marbox and signals for RNP binding are critical for transcriptional activation, presumably to align MarA with RNP. Introduction Transcriptional activation of bacterial promoters is thought to involve the binding of activator to DNA and interaction of activator with RNA polymerase holoenzyme (RNP) to recruit the RNP and/or to enhance a subsequent step of Received 26 March, 1999; revised 15 June, 1999; accepted 22 July, 1999. *For correspondence. E-mail [email protected]; Tel. (1) 301 496 5466; Fax (1) 301 496 0201. Q 1999 Blackwell Science Ltd

transcription (Ryu et al., 1994; Ptashne and Gann, 1997). In many cases, the activator binding sites are palindromic and bind tightly to dimeric activators. In a wellstudied case, activation by cyclic AMP receptor binding protein (CAP) involves interaction of CAP surfaces (AR-1 and -2) with subunits of RNP (for review, see Busby and Ebright, 1997). Activation at sites upstream of the 35 signal (class I) involves contacts between AR-1 and the carboxy-terminal domain of the a subunit of RNP (a-CTD). Activation at sites overlapping the 35 RNP signal (class II) involves contacts between AR-2 and the N-terminal domain of a (a-NTD). Transcriptional activation in the Mar system is interesting for several reasons. First, three independently regulated but homologous proteins, MarA, SoxS and Rob, activate a common set of promoters which results in multiple antibiotic resistance, superoxide resistance and organic solvent tolerance (for review, see Alekshun and Levy, 1997). Although these promoters are not stimulated to the same extent by all three activators (Wu and Weiss, 1992; Ariza et al., 1995; R. G. Martin, W. K. Gillette and J. L. Rosner, submitted), they are suf®ciently similar to be thought of as a single regulon (referred to here as the mar regulon) with three activators. marA expression is induced by salicylate and other phenolics (Cohen et al., 1993; Sulavik et al., 1995); soxS by paraquat and other superoxide-generating agents (Nunoshiba et al., 1992); rob has a high basal level of expression but for unknown reasons has little effect unless further overexpressed (Skarstad et al., 1993; Ariza et al., 1995). Second, each regulon promoter has a MarA /SoxS/Rob binding site, referred to here as the `marbox', many of which have been identi®ed by footprint analysis (Fawcett and Wolf, 1994; Li and Demple, 1994, 1996). Like CAP, MarA, SoxS and Rob are `ambidextrous' (Jair et al., 1995, 1996a, 1996b): when the marbox is located upstream of the 35 hexamer for RNP binding (as in the fpr and zwf promoters), activation requires interaction with a-CTD (class I); when the marbox overlaps the 35 hexamer (as in the fumC, micF and nfo promoters), aCTD is not required for activation (class II). Interestingly, the mar promoter itself has a marbox and is a member of the regulon (Miller et al., 1994; Martin et al., 1996).

432

R. G. Martin, W. K. Gillette, S. Rhee and J. L. Rosner

Table 1. Alignment of marboxes from mar/sox/rob regulon promoters. A. Comparison of mar (F-orientation) and mar (B-orientation) with two previous consensus determinations and with micF.a

±72c ±51 ±53

A A G A A

Y n A C T

n n T A G

G G T G C

C C T C C

A A A A A

B. Marbox sequences of ±63 A T G G C A ±55 A C T G C A ±47 A a G G C t ±53 A T G c C A ±51 g a G G C A ±63 A T G G C A ±61 A T C G C A

Y C G C C

n n C T G

Class C G C A C A C G C T C A C G

C. Marbox sequences of class A T A G C A t T ±56 A T G G C A C G ±51 A C G a C A C G ±51 A C A G C A C T ±46 A T C G C A t A A T C G C A C G ±52 A a A G C A g A ±49 A C G G C A t T A T G G C A C G A Y n G C A C n 15i 13 14 16 15 12

R n A G T

R n A A T

n n A A T

n n A T T

I promotersd A A A A C T G T A T C G T T T T A A C G A A T C G g T G

R n C G G

n n G T C

Y C T C T

A A G A A

n A G A A

n n C A A

n n A A T

n n T 53 C 32 C 72

Consensus Consensus mar micF mar

Fawcett and Wolf (1995)b Li and Demple (1996) Native orientation Native orientation Inverted orientation

A G A G G t G

C C T C T g a

C T C T T T T

A A A A A c A

A c A A A A A

A A A A A A g

C T T T T T C

A G C C A T G

acrAB e fldA fpr f mar poxB ribA zwf

(B) (B) (B) (B) (B) (B) (F)

II and uncharacterized promoters T T T A t C C A t A a A A A G A C C A A A C T T T C A T T A A g a G A A T G T C A A A a A A C C A C T A c A T A T C T G T a t A c T A A C T G T a A A A C G A T A A T C A t t T T A A C G C C A A c C n W n n R Y Y A A A Y 15 14 14 14 14 12 13 13

G A T C C T G T T n

82 74 66 72 70 82 42

37 32 32 27 33 30

(?) araI1 g fumC (F) inaA (F) micF (F) nfo (F) g (?) oriC pqi-5 (F) sodA (F) (F) tolC h Consensus (this work)

Miss [0] [1] [2] [1] [2] [3] [3]

[4] [0] [3] [1] [2] [2] [3] [3] [1]

Ftprt

Bind

Yes Yes Yes

Yes

Yes Yes

Yes

Yes Yes

Yes

Yes Yes Yes Yes Yes

Yes

Fnxn Yes Yes Yes Yes Yes Yes

Yes Yes Yes Yes

Yes Yes

a. Bold letters indicate mar marbox nucleotides that match the native micF marbox sequence when aligned in the native and inverted orientations. b. Abbreviations: R, A or G; Y, C or T; W, A or T. c. Numbers reflect position relative to TSS. d. Nucleotides that do not match the consensus sequence below are in lower case letters. F and B in parentheses refer to whether the native marbox is found in the F- or B-orientation. If naturally in the B-orientation, that sequence was inverted so as to align it with sequences that are naturally in the F-orientation such as micF. Compare with sequences in Fig. 2. Abbreviations: Miss, mismatch totals are in square brackets; Ftprt, footprint studies reported; Bind, binding to 20 bp fragments reported here; Fnxn, 20 bp marbox function seen in hybrid with micF promoter reported here. References: acrAB, Ma et al. (1993); araI1, Li and Demple (1996); fldA, Zeng et al. (1999); fpr, fumC, micF, nfo, sodA, zwf, Jair et al. (1996b); mar, Alekshun and Levy (1997); Sulavik et al. (1997); oriC, Skarstad et al. (1993); poxB, Chang et al. (1994); pqi, Koh and Roe (1996); ribA, Koh et al. (1996); Koh et al. (1999); tolC, Aono et al. (1998). e. TSS determined by A. Ball and H. Nikaido (personal communication). f. The numbering is relative to the prominent TSS that we have observed (data not shown); a TSS one base further downstream has also been observed (Jair et al., 1996b). g. The promoter controlled by this marbox is not known, so no orientation can be assigned. h. Relation to TSS is not known but marbox is 73 bp upstream and in F-orientation relative to the tolC structural gene. i. The number of sequences (out of 16) matching the consensus at the indicated position are shown.

Third, MarA, SoxS and Rob belong to a subfamily of the AraC family of DNA-binding proteins (Gallegos et al., 1997). They are unique in that they exist in solution and bind (and bend) DNA as monomers (Fawcett and Wolf, 1994, 1995; Jair et al., 1995, 1996a, 1996b). Recently, we have solved the structure of a MarA±marbox cocrystal, providing details of how this AraC homologue binds DNA (Rhee et al., 1998). In brief, two helix±turn±helix regions of the MarA monomer were found to contact bases in adjacent segments of the major groove of the marbox DNA. Although this has helped us to understand the basic design of MarA and its homologues, it has not furthered the de®nition of the marbox appreciably because the majority of the MarA±marbox DNA contacts are due to van der Waals interactions (see Discussion). To analyse the role of the marbox in transcriptional activation, it was important to develop a better de®nition

of the marbox. In spite of the DNase I protection studies (see Table 1 for references) and the MarA±marbox crystal structure, the de®nition of the marbox remained problematic because of its degeneracy, asymmetry and seemingly variable location with respect to transcription start sites (TSS) and RNP binding sites (Fawcett and Wolf, 1995; Li and Demple, 1996). Furthermore, the binding constants for different marboxes range from nanomolar to micromolar concentrations (Li and Demple, 1996; R. G. Martin, W. K. Gillette and J. L. Rosner, submitted). In this paper, we demonstrate: (i) that 20 bp naturally occurring marbox sequences can be identi®ed by DNA mobility assays; (ii) that these sequences can be aligned with sequences identi®ed by other methods to provide an improved consensus sequence based on 16 marboxes; (iii) that the marbox sequences can be shown to be Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 431±441

Marbox structural requirements

433

Fig. 1. Binding of MarA and the indicated 20 bp fragments covering the mar and micF marbox regions. The amounts of unbound and MarA-complexed [ 32P]-DNA in the gels were quanti®ed by phosphorimager analysis and used to derive the K Ds reported in the text. The numbers to the left and right of the sequences show the 58 and 38 positions relative to the transcription start site (TSS).

functional in a hybrid promoter system; (iv) that they are asymmetric, i.e. functional in only one orientation at a given site; and (v) that the functional orientation is dependent on the distance of the marbox to the RNP binding sites. This suggests that the orientation and spacing of the marboxes are critical for aligning the activators with RNP.

Results Identi®cation of minimal MarA binding sequences in promoters of the mar/sox/rob regulon and indication of inverted orientations Two highly degenerate consensus sequences have been proposed (Table 1A) for the binding site of MarA, SoxS and Rob in the promoters of the mar/sox/rob regulon (Fawcett and Wolf, 1995; Li and Demple, 1996). These sequences were based on footprint analyses of the small number of sites available at the time. To de®ne better the marbox, we have studied marboxes from a variety of promoters with respect to their binding of activators. The mar promoter marbox was studied ®rst because it bound MarA tighter than other marboxes: when present on a 135 bp fragment, the K D was 20 nM (Martin et al., 1996). Fragments as short as 20 bp bound MarA with a K D of 130 nM, but this increased sharply for 19 bp or smaller fragments (data not shown). We therefore prepared a Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 431±441

permuted set of 20-mers, corresponding to regions footprinted by MarA (Martin et al., 1996), beginning at positions 76 to 70 [relative to the transcription start site (TSS) of the mar promoter] and analysed their af®nities for MarA by gel mobility. Representative data are shown in Fig. 1; data from more extensive experiments are summarized in Table 2. This places the centre of the optimal mar 20-mer at 62.5, in agreement with a previous estimate from footprinting studies (Martin et al., 1996). Similar analyses (Table 2) were carried out for marbox sequences from the micF, fumC and zwf promoters, spanning the regions shown to be protected by SoxS from DNase I (Fawcett and Wolf, 1995; Li and Demple, 1996). The data suggest a polarity to the mar binding sequence that is opposite that of the others: the mar fragments that bound tightly were downstream of a fragment that bound poorly, whereas the reverse was true for the micF, fumC and zwf series. When the sequences of the optimal fragments for native micF and native mar are compared, only four bases match (Table 1A). However, if one of the sequences is inverted, 10 are identical. [The inversion of the mar sequence relative to that of micF had been predicted using the SEQUENCE WALKERS program (Schneider, 1997; T. D. Schneider and R. E. Wolf, Jr, personal communication).] Similarly, the sequence of the optimal mar binding fragment shows little similarity to the previously suggested consensus sequences: one match to the consensus of Fawcett and Wolf (1995) and four to

434


Table 2. Binding of MarA to permuted series of 20 bp fragments from several marboxes. Source of fragments micF

mar Fragment start 76 75 74 73 72 71 70

K D (nM) No binding No binding No binding 500 130 140 No binding

Fragment start

53 52 51 50 49

K D (nM)

No binding 150 130 500 No binding

fumC Fragment start

58 57 56 55 54

zwf K D (nM)

No binding 200 150 1300 No binding

Fragment start

63 62 61 60 59

K D (nM)

No binding 1500 1500 >1500 No binding

The upstream end of the 20 bp fragments tested for MarA binding by bandshift experiments is indicated by its distance from the TSS of the promoter. `No binding' means that no bandshift was detected. The K Ds were derived from multiple experiments using multiple dilutions of purified MarA.

that of Li and Demple (1996) (Table 1A). In contrast, the inverted mar sequence has six and eight matches to the abovementioned consensuses respectively. For clarity, we henceforth refer to the orientation of native marboxes that align with the native micF marbox (as shown in Table 1) as being in the F (forward)-orientation. We refer to all those native marboxes that need to be inverted to align them with the native micF marbox as being in the B (backward)-orientation. Thus, the native mar marbox is in the B-orientation, whereas micF, fumC and zwf are in the F-orientation. When present on large fragments (200±300 bp), the marboxes from the fpr and sodA promoters bound MarA with K Ds of 250 nM compared with 20 nM for the mar marbox (R. G. Martin, W. K. Gillette and J. L. Rosner, submitted). When similar experiments were carried out with various 20 bp fragments from the marbox regions of fpr and sodA (Fawcett and Wolf, 1994; Jair et al., 1996b; Li and Demple, 1996), no MarA binding was detected. Nonetheless, by aligning the footprint sequences of oriC (Skarstad et al., 1993), sodA, nfo (Jair et al., 1996b; Li and Demple, 1996) and pqi (Koh and Roe, 1996) in the Forientation and fpr in the B-orientation so as to obtain the maximum sequence identity with the optimal 20-mer sequences for mar, micF, fumC and zwf, we tentatively identi®ed the MarA binding sites in these sequences (Table 1). The validity of these assignments is demonstrated below. For the inaA promoter (White et al., 1992; Rosner and Slonczewski, 1994; Van Dyk et al., 1998), multiple attempts to de®ne the MarA binding region by footprinting were unsuccessful. We therefore mapped the transcription start site (TSS) of the inaA promoter by primer extension of isolated mRNA and found it to be located at position 10036 of section 203/400 (Blattner et al., 1997) of the GenBank Escherichia coli sequence (R. G. Martin, W. K. Gillette and J. L. Rosner, unpublished). To identify the inaA binding site in vivo, we constructed a series of 17 overlapping, single-copy inaA ::lacZ promoter deletions

and measured their activation by MarA or SoxS. The results demonstrate that stimulation of the inaA promoter by MarA and SoxS in vivo requires only sequences from 51 to 3 (data not shown). Although a series of 20 bp fragments starting at position 53 showed no binding to MarA by gel retardation (data not shown), we could readily align the inaA sequence from 51 to 32 in the F-orientation with the other optimal 20-mer sequences (Table 1). Determination of the orientations of the MarA binding sites in the promoters of the mar regulon As we have discerned binding sites with two possible orientations, we tested whether the orientation affected function by inverting the binding sites within the natural zwf and micF promoters. For zwf, the promoter fragment began at 61 with the 20 bp marbox shown in Table 1, or its inverted sequence, followed by the normal sequences of the zwf promoter from 41 to 3. For micF, the MarA binding sequence from 51 to 32 (Table 1), or its inverted sequence, was followed by the normal micF promoter sequences from±31 to 3. Because the binding site for MarA overlaps the 35 hexamer of micF, the inverted site alters the 35 hexamer sequence (AAAACA becomes ACTGTA) and reduces the basal level of expression considerably. Nevertheless, the TSS determined for the in vitro transcript was the same as that found for the normal micF promoter (data not shown). These fragments were cloned in plasmid pRS551, resulting in transcriptional fusions of each to lacZ. The b-galactosidase activities show that only when its marbox is in the F-orientation is the zwf promoter activated by MarA or SoxS (Table 3, top rows). Similarly, the micF promoter is activated by MarA or SoxS only when the micF marbox is in the F-orientation (Table 3, rows 10 and 11). Thus, the MarA /SoxS binding sites have functional and non-functional orientations at a particular promoter. If the binding sites of the mar regulon promoters were correctly identi®ed in Table 1 and interchangeable, it Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 431±441

Marbox structural requirements Promoter Core

Marbox

zwf

zwf micF fumC mar

micF

fpr micF zwf fumC inaA sodA mar fpr

inaA

acrAB d araI1 d fldA poxB d tolC d inaA

b-Galactosidase Orientationb

Uninduced (Miller units)

F B F B F B F B F F B F B F B F B F B F B F B F F F F F F

710 900 1900 580 890 880 710 1100 200 340 1.2 47 12 170 3.2 13 7.1 21 4.9 75 29 28 0.33 1000 26 81 320 550 29

Fold inductionc SAL PQ 2.1 1.3 2.7 0.9 2.6 1.1 3.5 0.9 2.0 6.9 0.7 4.3 1.5 6.4 1.7 2.8 1.8 1.6 1.5 10 1.2 1.3 1.8 ND ND 1.4 2.0 ND 6.9

435

Table 3. Effects of binding site orientation on inducibility of hybrid promoter::lacZ fusion activity in a high copy number plasmid.a

4.3 1.1 2.2 0.9 3.9 1.1 2.7 0.9 4.1 4.5 0.7 21 1.2 12 0.7 2.3 1.0 1.9 0.9 6.4 0.7 31 1.2 3.4 4.4 16 5.1 6.1 7.2

a. Hybrid promoters consisting of the indicated 20 bp marbox, the indicated core promoter and the lacZ fusion were constructed in plasmid pRS551 and transformed into strain RA4468 (rob ::kan) as described in Experimental procedures. These derivatives were exposed for 1 h at 328C to salicylate (SAL), to induce MarA, or to paraquat (PQ), to induce SoxS, and assayed for b-galactosidase. b. F, denotes the forward orientation; B, the backward orientation. c. The fold induction is the b-galactosidase activity in the presence of either SAL or PQ divided by the uninduced control. d. Activation was measured in strain DH5a.

should be possible to determine the functional orientation of other regulon binding sites by substituting the binding sequence of the tested promoter for the binding sequence present in zwf or micF. For example, if the binding sites for mar and fpr promoters are naturally in the B-orientation, as suggested, it might be necessary to invert these to the F-orientation for hybrids with zwf or micF core promoters to function. Accordingly, a series of hybrid promoters fused to lacZ was constructed in which the 20 bp binding sites from various regulon promoters, oriented in either direction, replaced the resident binding sites in the zwf and micF core promoters. Wherever the insertions altered the micF 35 hexamer, the TSS was mapped in vitro and found to be the same as the native micF TSS (data not shown). When SoxS was induced by treatment with paraquat (Table 3), the hybrid promoters were activated from twoto 31-fold if the binding site was present in the F-orientation but not at all if in the B-orientation. When MarA was Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 431±441

induced by treatment with salicylate, the stimulation was smaller, especially in the micF core promoter constructs. Nevertheless, clear responses were seen in hybrids consisting of the micF core promoter with binding sites from zwf, fumC and mar in the F-orientation but not in the Borientation. Thus, activation of the regulon promoters requires the proper orientation of the binding sites. For the marboxes from the mar and fpr sites to function in the micF or zwf promoters, they had to be inverted from their native B-orientations to the F-orientations. In contrast, when substituting a heterologous marbox for the native marbox of the mar promoter, the B-orientation is required for function (R. G. Martin, W. K. Gillette and J. L. Rosner, unpublished). With the knowledge that the binding sites exist in two orientations, we aligned 16 MarA /SoxS/Rob binding sites and sequences implicated in binding by either footprint or activation data to develop a new consensus binding sequence (Table 1, bottom row). We con®rmed that

436


Table 4. Transcriptional activation in vivo as a function of spacing between the marbox and 35 signal in the mar promoter. b-Galactosidase (Miller units) Spacing 3 2 1 0 1 2 3

a

(19 bp) (18 bp) (17 bp) (16 bp) (15 bp) (14 bp) (13 bp)

Strain N8452b 51 68 130 130 140 180 140

Strain N8452 /p37 35 95 330 640 670 400 160

Ratioc 0.7 1.4 2.5 5.0 4.8 2.2 1.1

a. The spacing between the binding site and the 35 signal in the transcriptional fusions was increased by 1±3 bp by the addition of T, TT or TTC, respectively, between positions 52 and 53 and decreased by 1±3 bp by deletion of bp 52, 52 and 51, or 52, 51 and 50, respectively. b. b-Galactosidase was assayed in Dmar rob ::kan cells (N8452) containing single-copy mar ::lacZ transcriptional fusions and in derivatives transformed with the MarA-overproducing plasmid p37. c. The extent of transcriptional activation is indicated by the ratio of activity in the N8452/p37-containing cells divided by that in the corresponding N8452 strain.

the potential marboxes from acrAB, araI1, ¯dA, poxB and tolC promoter regions are active when inserted in the Forientation in a hybrid with micF (Table 3). Interestingly, the marboxes of ¯dA, fpr, mar and ribA, in which the Borientation of the marbox was shown to be functional, are centred at least 52 bp upstream of the TSS, whereas those in the F-orientation occur downstream (see Fig. 3). Spatial relationship of MarA to RNP at the mar promoter The marboxes with the B-orientation are expected to be class I promoters and to enable the activator to interact with RNP, presumably via the a-CTD. We previously reported that the ability of MarA to stimulate the mar promoter was lost when the normal spacing between the end of the binding site and the 35 signal was increased by 5 bp from 16 to 21 bp (Martin and Rosner, 1997). We studied this in more detail by altering the normal spacing between the marbox and the core promoter by the addition or deletion of 1, 2 or 3 bp. Optimal activation of the mar promoter by MarA occurred when the spacing was the normal 16 or 15 bp and diminished progressively as the spacing was increased or decreased (Table 4). Thus, there are narrow limits to the spacing that permits a functional relationship between activator and RNP.

Discussion

that they can interact. For s70 and s38 promoters, the location of RNP on the DNA is determined by the 10 and 35 hexamers. The locations of MarA, SoxS and Rob on DNA have been deduced primarily by DNase I footprinting studies but, as the sequences are degenerate, a detailed de®nition of the marboxes has not been available. Here, in vitro studies were used to de®ne 20 bp marboxes from the fumC, mar, micF and zwf promoters that gave maximum binding (Fig. 1). Deletion analysis was used to delimit the marbox for inaA because activator binding was undetectable. By constructing hybrid promoters, we have shown in vivo that the 20 bp marboxes from the native acrAB, araI1, ¯dA, fpr, mar and poxB promoters are inverted (B-orientation) relative to that of micF, whereas those of fumC, inaA, sodA, tolC and zwf have the same orientation as that of micF (F-orientation). We deduced that the ribA marbox is also in the B-orientation from its location upstream of the TSS. This, and the inclusion of three other footprinted marboxes (nfo, pqi-5 and oriC ), has enabled us to compile an improved consensus sequence for the MarA, SoxS and Rob binding sites: AYnGCACnnWnnRYYAAAYn (Table 1, bottom row). In spite of the new data, it is clear that the sequence is highly degenerate. Orientation and phasing of class II marboxes In keeping with the asymmetry of the binding sites and the binding of MarA as a monomer, only one orientation of the sites was found to be functional at a given location on a promoter. Nevertheless, the binding sites are interchangeable provided they have the proper orientation and distance from the RNP site. For the six class II promoters, the F-orientation is functional. For several of these promoters, transcriptional activation does not require contact of MarA, SoxS or Rob with a-CTD (Jair et al., 1995, 1996a, 1996b) or with s-CTD (Jair et al., 1996a). Interestingly, these binding sites, which overlap the 35 hexamers, are centred 36.5, 39.5, 41.5, 42.5 and 46.5 bp distant from the TSS, the traditional way of describing binding site position on a promoter (Fig. 2A). This would not seem to position all the activators on the same side of the DNA helix relative to the TSS. However, when the distance from these binding sites to the presumptive 10 hexamer is considered, they are seen to be separated by either 18 or 19 bp (Fig. 2A). Thus, these marboxes are on the same side of the helix relative to the 10 hexamer and suggest that the relationship of marbox to 10 hexamer, and not to the TSS, is important. How MarA and RNP interact at these sites remains to be explored.

Marbox consensus sequence Prokaryotic transcriptional activation generally requires that RNP and activator be aligned at the promoter so

Orientation and phasing of class I marboxes Similarly, the class I binding sites do not have the same Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 431±441


437

Fig. 2. Sequences of mar regulon promoters showing the relationship of the marbox to putative 35 and 10 RNP signals and TSS (shown in bold). The assignment of 10 and 35 signals are supported by deletion analysis for mar (Martin and Rosner, 1995), but are only inferred for the others. Orientation of the native marbox is indicated by the heavy arrow inside the box (leftward backward; rightward forward). The number in the middle of the heavy arrow represents the centre of the marbox relative to the TSS. A. Class II promoters; 35 signals not indicated. B. Class I promoters.

helical phasing when viewed relative to their TSS. However, altering the spacing between the mar binding site and the 35 hexamer by inserting or deleting only two or three bases severely reduced transcriptional activation, indicating the importance of phasing (Table 4). This can be resolved by considering the distance from the marbox Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 431±441

to the 35 hexamer as crucial for alignment with RNP at these class I marboxes. This distance is 15 bp for fpr and 16 bp for mar and ¯dA (Fig. 2B). Reducing the distance from 16 to 15 bp in the natural mar promoter had only marginal effects (Table 4). Similarly, the spacing between the marboxes and putative 35 hexamers of

438


Fig. 3. Models of MarA bound to DNA. The surface of MarA, derived by X-ray crystallography [GRASP PROGRAM (Nicholls et al., 1991; Rhee et al., 1998)], bound to B-form DNA is depicted as it might exist when binding the mar promoter (left) or the zwf promoter (right). For the mar promoter, MarA is bound to a marbox in the B-orientation and displaced by 16 bp from the 35 hexamer (green arrowhead at the bottom of the ®gure). For the zwf promoter, MarA is bound to a marbox in the F-orientation and displaced by 7 bp. D18 and D22 indicate aspartic acid residues on the surface of the protein. The distances to the residues from the 35 hexamer are indicated.

acrAB and ribA promoters are 26 and 27 bp respectively (equivalent to one additional turn of the helix). Thus, the activators of the acrAB, ¯dA, fpr, mar and ribA promoters lie on one face of the DNA relative to RNP bound at the 35 hexamer. After submission of this article, Roe and colleagues (Koh et al., 1999) reported physical and

genetic studies showing that the ribA marbox is indeed in a reversed orientation (B-orientation, in our nomenclature). Curiously, the marbox of poxB, an RpoS-stimulated promoter (Chang et al., 1994), is separated by only 14 bp from the 35 signal, a spacing that severely reduced but did not Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 431±441


439

abolish stimulation of the mar promoter (Table 4). Whether this re¯ects a peculiarity of this promoter or is related to the dependence on RpoS is currently under investigation. Although the B-orientation is functional for class I marboxes 15 or more bp upstream of the 35 hexamer, the F-orientation is functional for the zwf class I marbox which lies 7 bp upstream of the 35 hexamer (Table 3; Wood et al., 1999). Thus, the activators would seem to have a very different relationship to RNP when bound to the zwf promoter than when bound to the other class I promoters. Whereas it may be the case that different contacts with RNP are used for the two situations, it is also possible that the different orientations and spacings of the marboxes permits the ¯exible a-CTD to contact the same residues on the back surface of MarA. We note that a negatively charged patch of amino acids on the surface of MarA is nearly equidistant from the upstream end of the 35 hexamer whether MarA is in the B-orientation and 16 bp from the 35 hexamer or is in the F-orientation and 7 bp away from the 35 hexamer (Fig. 3). Preliminary alanine scanning data suggests that amino acids in this region are important for the activation of mar and zwf promoters (W. K. Gillette, J. L. Rosner and R. G. Martin, unpublished).

13±15 of the consensus sequence respectively). Thr-93 makes H-bond contacts with bases at positions 66 and, via water, at 67 (14 and 15 of the consensus). Of these bases, a G occurs at position 3 in only 9 of 16 marboxes. Li and Demple (1996) found that changing single bases in the micF binding site sequence, corresponding to consensus positions 4 ±7 (GCAC), profoundly diminished SoxS binding; changes at consensus positions 15 and 16 (CA) had lesser effects. Nonetheless, the mar promoter marbox, one of the tightest binding marboxes in vitro, has a non-consensus C at position 4, perhaps because the tridentate Arg-46 can H-bond with either a G or C at this position. van der Waals contacts are made between Trp-42 and Gln-45 of MarA and bases at consensus positions C-5, A-6 and C-7. Interestingly, the presence of a T/A pair at consensus position 10 (15 out of 16), where no contacts with MarA were seen, may facilitate the distortion of the DNA. A similar analysis holds for the interaction of the 38 half of the binding site with MarA. The emerging view is that, although a few single sites may be very important, binding is due to overall shape complementarity of DNA and activator (Rhee et al., 1998) and it is the sum of many interactions that determines the precise binding to a marbox.

The consensus sequence and the MarA±marbox structure

Experimental procedures

Comparison of the consensus sequence with the MarA± mar marbox cocrystal structure (Rhee et al., 1998) suggests that subtle differences may exist in how the activator interacts with each marbox. The only invariant base in the sample of 16 binding sites is the C at position 5 [counting from the left (58 end) in Table 1, bottom row]. In the crystal structure, C-5 makes a van der Waals contact with Trp-42 of MarA. The complement (T) of the nearly invariant A-6 base (15 out of 16) makes van der Waals contacts with both Trp-42 and Gln-45. The complement () of A-16 is well conserved (14 out of 16) but it too makes van der Waals contacts with Thr-95 and Gln-92. However, the nearly invariant A-1 (15 out of 16) makes no contact with MarA and does not appear to be important for MarA recognition. The A-1 from the micF, fpr and zwf marboxes can be replaced with a G, C or T with no loss of activity in vivo (R. G. Martin and J. L. Rosner, unpublished; K. Grif®th and R. E. Wolf Jr, personal communication). Why A-1 is conserved is unknown. Along with Trp-42 and Gln-45, Arg-46, Thr-93 and Arg96 are conserved in MarA, SoxS and Rob. Arg-46 makes direct contacts via hydrogen bonds to bases of the mar binding site located at positions 55 and 56, and Arg-96 makes H-bond contacts with bases at 65 and 66, and, via water, at 67 (corresponding to positions 3, 4 and Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 431±441

Marbox and promoter constructions Oligonucleotides were synthesized on a DNA /RNA Applied Biosciences Synthesizer and used in PCR reactions, endlabelled with 32P or cloned by standard procedures (Sambrook et al., 1989). The promoter fragments used for binding studies and for fusion to lacZ were synthesized as complementary oligonucleotides with the following sequences for one strand: (i) 58-GGAATTC (to create an EcoRI site); followed by (ii) the marbox (see Table 1) (or the inverted marbox); followed by (iii) the core promoter into which the marbox was being transposed (position 31 to position 3 of micF and position 41 to position 3 of zwf); followed by (iv) GGATCCG-38 (to create a BamHI site). The complementary sequence was used for the other strand. Thus, the micF promoter with the F-orientation micF binding site was cloned as: GGAATTC ACAGCACTGA ATGTCAAAAC AAAACCT TCA CTCGCAACTA GAATAACTCC CGCT GGATCCG, and the micF promoter with the B-orientation zwf binding site was: GGAATTC CGCTTATCCA CCCGTGCGAT AAA ACCTTCA CTCGCAACTA GAATAACTCC CGCT GGAT CCG. These promoter fragments were digested with EcoRI and BamHI and inserted into similarly digested pRS551, thereby creating a transcriptional fusion with lacZ (Simons et al., 1987). The resulting plasmids were then transformed ®rst into DH5a competent cells (Gibco) selecting for AmpR and from there to the rob ::kan strain RA4468 (Ariza et al., 1995). Single-copy lysogens were prepared by growing lRS45 phage on the transformants and selecting KanR colonies in appropriate host strains GC4468 (wild type), N7840

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(Dmar ) or N8452 (Dmar, rob ::kan) as previously described (Martin and Rosner, 1997). Where indicated, these cells were then transformed with the MarA overexpressing plasmid p37 (Gambino et al., 1993). The various inaA ::lacZ constructs were prepared similarly. The fragments were prepared by PCR reactions using strain GC4468 (inaA) DNA as template and appropriate primers. The fragments were then cloned into pRS551 to make the lacZ fusions and transferred to lRS45 as above.

Growth of cells and b-galactosidase assays Cells were grown in Luria±Bertani (LB) broth, pH 7.5, at 328C, treated with 5 mM sodium salicylate or 50 mM paraquat for 1 h, and assayed for b-galactosidase (expressed as Miller units) as previously described (Miller, 1972; Martin and Rosner, 1997). All assays were carried out in duplicate and repeated at least twice; standard deviations were less than 20%.

DNA-binding assays MarA was puri®ed to homogeneity and the histidine tag removed as previously described (Jair et al., 1995). soxS was cloned in pET15b (Novagen) and transformed into Novagen strain B834(DE3). SoxS was overexpressed, puri®ed to homogeneity and the histidine tag removed by identical techniques. Gel mobility experiments were performed in 4% or 6% acrylamide gels in TAE buffer as previously described (Martin and Rosner, 1995). The dissociation constants (K D ) were determined from multiple experiments using multiple dilutions of activator.

Acknowledgements We thank Richard E. Wolf Jr and Thomas Schneider for discussions and for communicating information before publication; Hiroshi Nikaido and Gisela Storz for communicating information on ¯dA and acrAB respectively; and Philip Ross for advice on statistics.

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