Birth Rate Increases following Improved Rural Water Supply

April 2006 | Volume 3 | Issue 4 | e133 | e147 Development efforts in rural Africa over the last few decades have achieved improvements in living conditions and in health. It has been argued that when such changes occur, there will be a subsequent reduction in birth rates, and experience in other parts of the developing world has tended to bear out this prediction. However, birth rates in rural Africa remain high and the population continues to grow rapidly. The situation in Ethiopia provides an illustration; spiraling population growth and slow economic growth are widely considered to be the main factors that have fuelled this country’s repeated humanitarian crises. On the basis of current trends, it is predicted that Africa’s population will double in the next 50 years, but in many countries, the resources are currently not available to sustain such a level of growth, and increased human suffering may be the consequence. An important question, but one that is seldom discussed, is whether development programs in Africa fail to make sustainable improvements over the long term because they lead to unsustainable increases in population growth rate. This concern is addressed, however, in a paper by Gibson and Mace, who studied a rural development program that was intended to improve the lives of Ethiopian women. The researchers measured its impact on the health of women and children in eight villages included within the program, and also on the birth rate. The study involved a rural area where some villages had benefi ted from the provision of a tapped water supply. Previously, women had to walk long distances (up to 30 km) to fetch their families’ water in clay pots. The development program reduced the time they spent carrying water each day from about three hours to about 15 minutes. The researchers collected information over a four-year period, including for both villages where tapped water had been introduced and for others where it had not. In total, nearly 2,000 households were included. The nutritional status of the women and children (in terms of body mass index) was also measured. The researchers found that the availability of tapped water improved the survival of young children, although their nutritional status actually declined, and the birth rate increased. All this caused greater scarcity of resources within households. The incremental effects of small changes to energy balance caused by development can increase strain on the household by increasing birth rates. This fi nding highlights the importance of continuing to improve access to contraception, especially in rural areas.


INTRODUCTION
Bacteriophage l is a temperate phage which can enter one of two alternative developmental pathways, lytic or lysogenic, upon infection of its host, Escherichia coli (1,2). When the lysogenic pathway is chosen, phage DNA is incorporated into the E. coli genome, forming a prophage that can be maintained in this state for many cell generations. Stable maintenance of the prophage is achieved through the action of the phage-encoded repressor, the l repressor (CI), which both represses the lytic promoters, p L and p R , and stimulates transcription of its own gene from the p M promoter (3). The p R and p M promoters are divergently arranged with their start sites separated by only 82 bp. Both promoters are regulated by the binding of CI dimers to three related 17-bp sequences, O R 1, O R 2 and O R 3, located at À74 to À58, À50 to À34 and À27 to À11, respectively, with respect to the transcription start site at p M . A CI dimer bound at the highaffinity operator, O R 1, acts as a repressor of the p R promoter but also stabilizes the binding of a second CI dimer to a lower-affinity operator, O R 2, and the second dimer, in turn, interacts with RNA polymerase (RNAP) to stimulate transcription from p M above basal levels (3,4). This stimulation occurs at the isomerization step (k f ) in the transcription initiation pathway that leads to open complex formation (5,6). At higher concentrations, CI also binds to O R 3, thereby repressing p M (7).
Each CI monomer comprises an N-terminal DNAbinding domain (residues 1-92) and a C-terminal oligomerization domain (residues 132-236) connected by an interdomain linker known as the 'hinge' region (8). Detailed structural information is available for the isolated N-terminal and C-terminal domains (9)(10)(11)(12)(13). The oligomerization domain participates in dimerization of CI monomers and is also involved in weaker cooperative interactions between pairs of dimers bound to adjacent operator sites. The nature of both of these types of interaction have been elucidated by X-ray crystallography (12,13). It has also been shown that repressor tetramers repression of p R (13)(14)(15). The N-terminal domain of CI contains a DNA-binding helix-turn-helix motif which is responsible for operator recognition. In addition, residues exposed on the first helix (specifically E34 and D38) generate a negatively charged patch which, in the case of the downstream subunit of the CI dimer bound to O R 2, is involved in interactions with positively charged residues (R588, K593 and R596) on the surface of domain 4 of the RNAP s 70 subunit (s 4 ) during activation of p M (6,(16)(17)(18)(19)(20)(21)(22)(23). For this reason, CI is classified as a Class II activator, along with other activators which bind to sites overlapping the À35 region and, in most cases, activate transcription by contacting s 4 (22,24,25).
At many bacterial promoters, the C-terminal domain of the RNAP a subunit (aCTD) interacts with upstream promoter DNA, the RNAP s 70 subunit and/or transcription activator proteins (24,26). These interactions are mediated by determinants on the surface of aCTD and are facilitated by the presence of a flexible linker connecting aCTD to the N-terminal domain (27)(28)(29). For example, residue 265, and neigbouring residues, contribute to the 265 determinant, which is responsible for interactions with DNA (30)(31)(32)(33). Similarly, residue 261 and neighbouring residues contribute to the 261 determinant, that can contact s 4 (34)(35)(36), whereas the side chains of valine 287 and neighbouring residues form a surface-exposed patch, the 287 determinant, which interacts with an activatory surface, AR1, on CRP (cyclic AMP receptor protein) and with other activators (33,34,37,38).
Previously, we have shown that the rpoA341 mutation, leading to substitution of glutamate for lysine at position 271 within aCTD, decreases l prophage stability (39,40). This observation could be explained by a defective interaction between the mutant aCTD and the CI repressor at p M . Therefore, the aim of this work was to determine whether aCTD plays a role in CI-dependent activation of p M . Our results show that determinants on the surface of aCTD are required for fully efficient activation by CI. In addition, we demonstrate that the location of aCTD at p M is shifted further upstream in the presence of CI. These observations suggest that CI makes direct contact with aCTD at p M and that this interaction is important for transcription activation by CI.

Bacteriophage, plasmids and gene fusions
Bacteriophage lcI857S7 (44), which is unable to lyse E.coli cells unless the supF suppressor allele is present, was used for measuring prophage stability. For the expression of mutant rpoA alleles for the aCTD alanine scan analysis, derivatives of plasmid pHTf1a, encoding a mutants with alanine substitutions at positions 255-271 and 302, and pREIIa, encoding a mutants with alanine substitutions at the remaining positions in aCTD, were used (27,30,37,(45)(46)(47). Plasmids pGW857 and pAClcI, both of which are p15A derivatives, were used to express the phage l cI gene. pGW857 encodes the thermolabile CI 857 protein under control of the lac promoter (48) and thereby allows for complete inactivation of repressor function by growth at 428C. Plasmid pAClcI was used to overexpress the wild-type cI gene from the lacUV5 promoter (49). For measuring the activity of the p M promoter, two p M -lacZ fusion plasmids were used: pAHA1, a pBR322-based replicon, and pTJSpM, an RK2-based replicon. To construct pAHA1, the wild-type p M promoter region (248 bp) was amplified by PCR using the l plasmid pKB2 (50) as a template, and the following primers: 5 0 -GCC GGA TCC CCA TCT TGT CTG C and 5 0 -TAT GCG TTG TTA GCT ATA GAC TCC TTA GTA C (35 cycles of the following program were performed: denaturation at 958C for 30 s, annealing at 55.48C for 30 s, extension at 728C for 30 s). The product of the amplification was digested with BamHI and cloned between the BamHI and SmaI sites upstream of the lacZ gene of pHG86 (51). To construct pTJSpM, the EcoRI-HindIII fragment containing the p M promoter was cut from plasmid pEM9-O R P (52) and used to replace the BamHI-EcoRI fragment of pTJSpI containing p I (53), following treatment of both the vector and the promoter fragment with T4 DNA polymerase. The p M promoter present in pEM9-O R P (and pTJSpM) contains the wildtype O R 1 and O R 2 operators, but O R 3 is inactivated by multiple mutations (TACAGCTGCAAGGGATA). These changes (underlined) abolish CI binding but do not alter the À35 or À10 sequences of the p M promoter. pJMH1 is a pSC101 derivative carrying the lacI q and kanamycin resistance genes (39). pRLGpMmut was constructed by amplifying a DNA fragment containing the phage l p M promoter using primers 5 0 -GCC GAA TTC GTA CAT GCA ACC ATT ATC-3 0 and 5 0 -TTG TAA GCT TAC GTT AAA TCT ATC ACC ACA AGG G-3 0 (35 cycles of the following program were performed: denaturation at 958C for 20 s, annealing at 508C for 30 s, extension at 728C for 60 s). This fragment was ligated between the HindIII and EcoRI sites of pRLG770 (54). The second primer introduces a G to A point mutation at À18 (underlined) which reduces binding of CI to O R 3 and consequent repression of p M (55).

Measurement of the effect of mutant rpoA alleles on CI-dependent activation in vivo
For the alanine scanning experiment (merodipoid), expression of wild-type cI from pAClcI, and mutated rpoA alleles from pHTf1a and pREIIa derivatives, was simultaneously induced by addition of IPTG (0.1 mM final concentration) to cultures of WAM106 harbouring pJMH1 and pTJSpM growing at 378C. The b-galactosidase activity was measured 1 h later. To assess the effect of haploid rpoA alleles on CI-dependent activation of p M , strains harbouring chromosomal mutant rpoA alleles were transformed with pGW857 and pAHA1, and cultures were grown at 438C to OD 575 ¼ 0.2 [the cI857(ts) gene product is inactive under these conditions and b-galactosidase activity is very similar to that measured in cells devoid of pGW857; data not shown] whereupon IPTG was added (0.05 mM final concentration) and the culture was immediately shifted to 308C. Following incubation at this temperature for 1 h the b-galactosidase activity was measured. This induction regime minimizes problems due to CI occupancy of O R 3 present on pAHA1 (data not shown).

Measurement of b-galactosidase activity
The activity of b-galactosidase in bacterial cells was measured according to Miller (56). Since we used a multicopy lacZ fusion, the b-galactosidase activities were calculated per plasmid copy number, estimated as described previously (57), to compensate for any possible copy number variation between strains. For the alanine scanning experiment, bacteria were grown at 378C to OD 578 ¼ 0.2, induced with 0.1 mM IPTG and, following further incubation for 1 h, b-galactosidase assays were performed. Results presented are averages of at least three independent experiments and are shown with standard deviations.
Measurement of the efficiency of prophage maintenance l prophage maintenance in lysogenic E.coli strains was estimated by measuring the efficiency of spontaneous induction of a lcI857S7 prophage as described previously (40). Briefly, samples (5 ml) of exponential phase cultures (OD 578 0.2-0.5) of bacteria lysogenic for bacteriophage lcI857S7, growing at 308C, were withdrawn and shaken vigorously with chloroform (0.5 ml) for 1 min to release progeny phage. Following centrifugation, liberated phages were titrated on the suppressor strain, TAP90, at 378C. Other samples, withdrawn at the same time as those for phage titration, were centrifuged. Cell pellets were resuspended in 0.9% NaCl and used for titration of bacteria on LB agar at 308C. Finally, the number of phages yielded per bacterial cell was calculated.

Protein purification and reconstitution of RNA polymerase
Plasmid pT7lcISa109His6 (21) was used for overproduction of C-terminally His-tagged CI protein, which was purified as described previously (21). For the reconstitution of RNAP, inclusion bodies of RNAP b, b 0 and s 70 subunits from strains XL1-Blue (MKSe2), BL21(DE3)(pT7b 0 ) and BL21(DE3)(pLHN12s), respectively, were prepared as described previously (58). Histagged RNAP a subunits were prepared using plasmid pHTT7f1NHa (58). Derivatives of pHTT7f1NHa carrying mutant rpoA alleles were constructed by replacing the HindIII-BamHI fragment, which encodes aCTD and the interdomain linker, with the corresponding fragments from derivatives of pHTf1a and pREIIa encoding the appropriate alanine-substituted a mutants (see above) or from pLAW2phs (encoding a containing the K271E substitution) (39). Overexpression of the a subunits in strain BL21(DE3), purification of a by Ni 2þ -affinity chromatography and reconstitution into RNAP were performed essentially as described previously (30,58). Purification of a subunits with single cysteine residues, conjugation with Fe.BABE, and reconstitution into RNAP was performed as described by Lee et al. (59).

In vitro transcription
Single round in vitro transcription reactions were performed in a total volume of 20 ml in buffer containing 50 mM KCl, 40 mM Tris-HCl (pH 8.0), 10 mM MgCl 2 , 1 mM DTT, 100 mg/ml BSA and 30 ng linear template DNA. Template DNA containing the p M promoter was prepared by isolating the 1260-bp NdeI-EcoRI fragment from plasmid pRLGpMmut. The 1313-bp NdeI-PstI fragment from the same plasmid, containing the RNA I gene, served as the internal control. The binding reaction of CI (80 ng) to the DNA (30 ng) was carried out at 378C for 10 min, after which time in vitro reconstituted RNAP was added and the incubation continued for a further 10 min (this concentration of CI gave rise to $4-fold activation of p M in the presence of wild-type reconstituted RNAP (results not shown)). After the addition of nucleotides (CTP, GTP and ATP, each to a final concentration of 150 mM, UTP to 15 mM and 0.6 mCi [a-32 P]-UTP per reaction) and heparin to 50 mg/ml, the samples were incubated at 378C for 15 min and the reactions were stopped by the addition of an equal volume of 95% formamide containing 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol. The samples were separated by electrophoresis in 6% polyacrylamide gels containing 46% urea in TBE buffer. The gel was dried, and RNA bands were visualized and quantified, following background subtraction, using a PhosphorImager (Bio-Rad). Concentrations of RNAP, calibrated to give the same amount of transcription from the activator-independent RNA-I promoter, were: 46 nM wild-type RNAP, 34 nM RNAP aK271E, 54 nM RNAP aK271A, 13 nM RNAP D258A, 28 nM RNAP aE261A, 35 nM RNAP aR265A, 34 nM RNAP aV287A.

FeÁBABE-mediated hydroxyl radical footprinting
A 150-bp DNA fragment containing the l p M promoter was amplified from bacteriophage l DNA by PCR using primers 5 0 -GCT TTA AGC TTA CGT GCG TCC TCA AGC TGC-3 0 and 5 0 -CCT GAA TTC ATG CAA CCA TTA TCA CCG-3 0 , cleaved with HindIII and EcoRI and cloned into the vector pSR (60). A 220-bp AatII-HindIII fragment was purified from the resultant plasmid (pSRpM) and labelled at the HindIII end with [g-32 P]-ATP and T4 polynucleotide kinase. The FeÁBABEmediated DNA cleavage reactions were carried out in a reaction volume of 25 ml (5 mM MgCl 2 , 100 mM potassium glutamate, 40 mM HEPES pH 8.0, 50 mg/ml BSA, 10 mg/ml herring sperm DNA). Promoter DNA fragments were incubated with CI protein (250 nM final concentration) at 378C for 10 min. After 10 min, RNAP holoenzyme was added (600 nM final concentration) and incubated at 378C for 30 min. Complexes were then challenged with heparin (50 mg/ml final concentration) for 1 min at 378C then DNA cleavage was initiated by the addition of 3 mM sodium ascorbate and 3 mM hydrogen peroxide. The reactions were incubated for 10 min before being stopped by the addition of thiourea and EDTA to final concentrations of 7 mM and 45 mM, respectively. DNA was then extracted with phenol/chloroform, precipitated with ethanol and analysed by electrophoresis in a 6% denaturing polyacrylamide gel. The gels were calibrated with Maxam-Gilbert G þ A ladders and analysed using a PhosphorImager and Quantity One software (Bio-Rad).

Identification of aCTD determinants important for CI-dependent activation of p M
To identify whether amino acid side chains on aCTD are important for activation by CI, we used an alanine scanning approach, exploiting a set of plasmids encoding the RNAP a subunit in which residues 255-329 were each changed individually to alanine. This approach has been used to identify aCTD residues important for transcription activation mediated by a number of different activator proteins (34,37,38,41,53,(61)(62)(63). These plasmids were introduced into an E.coli rpoA þ strain carrying a p M -lacZ fusion plasmid and inducible CI function.
The results show that, under conditions promoting CI stimulation of p M , alanine substitutions at residues R255, P256, D258, E261, S266, N268, C269, L270, V287 and S299 in aCTD most strongly impaired the activity of p M (i.e. activity 80% of that afforded by plasmid-encoded wild-type a) ( Figure 1A). The location of these residues in the aCTD structure is shown in Figure 1B (the residues at positions 266, 270 and 299 are buried in the aCTD structure and are therefore not included in this figure). Most of them are located on one side of aCTD and create a patch on the surface of the domain, whereas V287 is located on the opposite side of aCTD.

Effect of substitutions in aCTD on CI-dependent activation of p M in vitro
To determine whether the effects of the alanine substitutions on in vivo p M activity are direct, we measured the efficiency of CI-mediated stimulation of p M in vitro, using run-off transcription assays. RNAP was reconstituted with the wild-type a subunit, and with some of the mutant a subunits giving rise to a significant decrease in p M promoter activity in vivo (i.e. a containing the 258A, 261A and 287A substitutions). To confirm that R265, within the aCTD DNA-binding determinant, does not play an important role in CI-dependent activation of p M , RNAP was also reconstituted with the R265A a subunit. In addition, due to our previous observation that the K271E substitution in a causes decreased prophage stability, we included RNAP reconstituted with the 271E and 271A a subunits in the analysis.
Our results are in general agreement with the in vivo results, i.e. the abundance of p M -derived transcripts was significantly decreased when RNAP was reconstituted with a containing the 258A, 261A and 287A substitutions, whereas the efficiency of transcription obtained using RNAP reconstituted with a harbouring the 265A substitution was comparable to that of wild-type RNAP ( Figure 2). Consistent with its effect on prophage stability, RNAP reconstituted with 271E a was significantly less active at the p M promoter in vitro. This was also the case with 271A a, although alanine substitution at this position does not exert a negative effect at p M in vivo ( Figure 1A). Under these conditions we observed $5-fold activation of transcription from p M in the rpoA þ host (Table 1), which compares favourably with previously reported induction ratios (19,20). However, in strains harbouring the mutant rpoA alleles, CI-dependent activation of p M was only 45-60% as efficient as in the wild-type strain, with the C269A substitution causing the most profound effect (Table 1). By way of comparison, the p M activity in the strain harbouring the rpoA341 allele, encoding the K271E substitution in a (39,40), was $55% as efficient as in the wild-type strain (Table 1). These results confirm the important roles played by the 261 and 287 determinants and the DNA-binding region of aCTD in CI-dependent activation at p M .

Effect of substitutions in
Prophage stability. As maintenance of a l prophage only requires CI function, we investigated whether substitutions within aCTD which impair CI-dependent activation of the p M promoter also impair l prophage maintenance. To do this, we compared the efficiency of spontaneous induction of a lcI857S7 prophage in hosts harbouring wild-type or mutant rpoA alleles on the chromosome. As expected, we found that alanine substitution at positions 261, 269, 271 and 287 in a resulted in a higher frequency of spontaneous induction of the l prophage relative to the wild-type host (3-8-fold increase, depending on the position of the substitution) ( Table 1). Consistent with the p M promoter activity measurements, the prophage was most unstable in the host carrying the rpoA269 allele. As shown previously, we measured a 5-fold increase in spontaneous induction of l prophages in the rpoA341 mutant relative to the wild-type (Table 1; 40). In support of the hypothesis that decreased prophage stability was due to decreased CI levels, overexpression of the cI gene from plasmid pAClcI resulted in equally efficient maintenance of the prophage in the wild-type and in all tested mutant strains (data not shown).

Location of the aCTD-DNA interactions at the p M promoter
To determine the location of aCTD at the p M promoter we exploited the DNA cleavage reagent, iron [S]-[p-bromoacetamidobenzyl] ethylenediaminetetraacetate (FeÁBABE), that can be attached to cysteine residues introduced at specific locations within aCTD (59,64,65). Thus, we derivatized aCTD with FeÁBABE by employing a functional a subunit in which cysteine was introduced at position 273, and used the derivatized product to reconstitute RNAP (53,59).
Analysis of DNA scission products following formation of the RNAP-FeÁBABE-p M complex revealed that, in the absence of CI, cleavages occur in clusters separated by 10-11 bp, with the strongest signals occurring near position À44 relative to the transcription start site (Figure 3). This is consistent with the fact that p M serves as a weak promoter in the absence of CI (66). The pattern of cleavages is similar to that found at other promoters that are active in the absence of transcription activators, such as rrnB P1 or CC(À61.5)-p12T (59), and suggests that one of the two a subunits binds to the first available minor groove upstream of the À35 region while the second aCTD binds to successive minor grooves (i.e. À54, À65 and À75, with À54 being the most favoured position) (Figure 3). This is in accordance with previously published results, which suggested that the a subunit contacts sequences upstream of p M in a sequence non-specific manner (67). In the presence of CI, the strongest signals were observed near position À54, which is located in the minor groove between two CI dimers bound to major grooves within O R 2 (À34 to À50) and O R 1 (À58 to À74) (68) (Figure 3). Therefore, binding of CI results in re-positioning of aCTD at the p M promoter.

DISCUSSION
The location of the stimulatory CI-binding site (O R 2) at p M (see Figure 3B) suggests that CI activates this promoter by a Class II-type mechanism (22,24,69). Consistent with this, it has been shown that a negatively charged patch on the surface of the CI DNA-binding domain, located in helix 1 of the HTH motif, stimulates transcription from p M through making contact with a positively charged patch on s 4 (23). In this report, we have demonstrated that determinants on aCTD also contribute to CI-dependent activation of p M . Alanine scanning analysis indicated that some of the surfaceexposed residues on aCTD which are required for efficient CI-dependent activation are located within or near the previously identified 261 determinant (i.e. R255, P256, D258, E261 and K271) and the 287 determinant (V287). These determinants are located on opposite sides of aCTD and have been shown to play roles in activator-dependent transcription at other promoters. It is intriguing that the 261 determinant is implicated in CI-dependent activation, as it has previously been shown to play a role only at Class I CRP-dependent promoters and at some UP element-dependent promoters, where it interacts with s 4 (34)(35)(36). At other Class II promoters, where aCTD is not in a position to interact with s 70 , the 261 determinant does not play a role in transcription activation (37). Our results with FeÁBABE-derivatized RNAP show that, in the presence of CI, aCTD is located close to position À54 at p M , i.e. between O R 1 and O R 2, and therefore is also not in a position to contact s. Therefore, the simplest explanation for our observations is that the 261 determinant is involved in contacts with CI. The 287 determinant has been shown to interact with CRP at Class I and Class II CRP-dependent promoters and there is evidence that it interacts with MelR at the pmelAB promoter (34,37,38). Our results suggest that CI is another activator that utilizes this determinant. The involvement of residues on opposite sides of aCTD in CI-dependent activation could occur if aCTD is sandwiched between the two CI dimers, as demonstrated by the FeÁBABE analysis, with each determinant  interacting with a different CI dimer. This is analogous to the situation at the artificial Class II promoter, ML(À74.5), which contains tandem CRP sites centred at À41.5 and À74.5. At ML(À74.5), one aCTD is recruited to the DNA between the two CRP-binding sites, whereas the other aCTD binds to DNA upstream of the CRP dimer bound at À74.5 (70). Furthermore, the 261 and 287 determinants of the aCTD sandwiched between the CRP dimers are likely to be aligned along the axis of the DNA, with the 287 determinant interacting with AR1 of the promoter-proximal CRP, as shown for the simple Class II CRP-dependent promoter CC(À41.5) (37,59). Although the location of the second aCTD at p M was not addressed in this investigation, one intriguing possibility is that, in a situation where O L (the CI operator overlapping the p L promoter) is also present, the second aCTD binds O L between the pair of CI dimers bound to the O L 1 and O L 2 sites. Our results also revealed that alanine substitution of amino acids S266, N268, C269, L270 and S299 impaired CI-dependent activation. These residues are located within or near the DNA-binding surface of aCTD (33,71) (although L270 does not participate directly in DNA binding, the side chain is buried within the structure of aCTD and therefore substitution by alanine may cause a conformational change in the DNA-binding region).
The DNA-binding determinant plays a role in UP element-dependent transcription initiation and at many activator-dependent promoters (24,30,34,37,53). Its involvement in CI-dependent transcription activation suggests that an interaction between aCTD and the promoter is important for CI-dependent activation. The results of the Fe.BABE analysis suggest that the important aCTD-DNA interaction is likely to be due to the aCTD positioned near À54. It is noteworthy that the side chain of R265, which plays an important role in DNA binding at many promoters, does not appear to be required for efficient CI-dependent activation. However, it has been shown previously that the contribution of this residue to DNA binding at some activator-dependent promoters is minimal (34). On the other hand, the broader Fe.BABE cleavage pattern that occurs at À54 in the presence of bound CI, in comparison to the more focussed cleavage at À44 in the absence of CI, may indicate that aCTD is not in intimate contact with the DNA when CI is present (i.e. aCTD may be interacting with CI 'off the DNA') or that the interaction of the DNA-binding determinant with the promoter is different to that which occurs at many other promoters. One possible reason for this is that, for steric reasons, aCTD may not be able to readily access the À54 region on the same side of the DNA as CI (Figure 4). Firstly, the diameter of aCTD (measured from the 261 determinant to the 287 determinant) is $25 Å . Although the distance between the two operators, O R 1 and O R 2, is $24 Å (based on a rise of a 3.4 Å per bp), the separation between the two CI dimers is likely to be less than this. This is due to the fact that the adenine tract between the two operator sites contains a static bend of the order of 188, which becomes further bent by 15-188 upon binding CI, in a large part due to untwisting of the DNA (13,(72)(73)(74). Access to the DNA between O R 1 and O R 2 may be further restricted by the cooperative interactions which occur between the C-terminal oligomerization domains of CI (12,13,73).
The other important observation from this investigation is that the location of aCTD at p M is different in the presence and absence of CI. In the absence of CI, one aCTD is located adjacent to s 70 at a site that overlaps O R 2. In the presence of CI, O R 2 is occupied by CI and aCTD is relocated to a DNA site located between O R 1 and O R 2 ( Figure 4). This observation, together with the analysis of a mutants, is consistent with a model in which activation of RNAP at p M is mainly the result of the interaction between CI bound at O R 2 and s 70 , as previously proposed (19)(20)(21)(22). The role of the aCTD-CI interaction may be to stabilize the interaction of aCTD with DNA upstream of O R 2, facilitating CI-dependent stimulation of the k f step.
CI is not the only Class II transcription activator to make contact with aCTD in addition to s 4 . Both MelR and CRP (at the galP1 promoter) also make a specific contact with aCTD, and this interaction contributes to the overall stimulatory activity of the regulatory protein (24,38,75,76). Other examples of so-called 'ambidextrous' activators include LuxR and the phage Mu Mor protein (76)(77)(78)(79). In such cases, aCTD binds to the first available minor groove upstream of the activator binding site, with a preference for binding to the same face of the DNA as RNAP (38,53). In the case of p M , the first available minor groove is located between the two CI dimers bound at O R 1 and O R 2. and Simon Dove for plasmid pT7lcISa109His6.
Conflict of interest statement. None declared. The most efficient cleavages induced by Fe.BABE tethered to aCTD occur around À44, with slightly less efficient cutting at À54. Thus, the promoterproximal aCTD is 'parked' at O R 2. (B). Proposed location of aCTD at p M in the presence of CI based on FeÁBABE analysis. In the presence of CI, aCTD bound at O R 2 is displaced to the À54 region, between O R 1 and O R 2, and is possibly sandwiched by the DNA-binding domains of the two CI dimers. Note that although aCTD is shown as contacting the DNA at À54 in the presence of CI, it is also possible that there is no direct contact between aCTD and this region of the DNA. The location of the other aCTD was not determined in this analysis.