We have investigated the role of the RNA polymerase α subunit during MelR-dependent activation of transcription at the Escherichia coli melAB promoter. To do this, we used a simplified melAB promoter derivative that is dependent on MelR binding at two 18 bp sites, located from position −34 to −51 and from position −54 to −71, upstream of the transcription start site. Results from experiments with hydroxyl radical footprinting, and with RNA polymerase, carrying α subunits that were tagged with a chemical nuclease, show that the C-terminal domains of the RNA polymerase α subunits are located near position −52 and near position −72 during transcription activation. We demonstrate that the C-terminal domain of the RNA polymerase α subunit is needed for open complex formation, and we describe two experiments showing that the RNA polymerase α subunit can interact with MelR. Finally, we used alanine scanning to identify determinants in the C-terminal domain of the RNA polymerase α subunit that are important for MelR-dependent activation of the melAB promoter.
The C-terminal domains of the bacterial RNA polymerase α subunits (αCTD) play a significant role in transcription activation at many promoters (Ebright and Busby, 1995; Gourse et al., 2000). Many activators interact directly with αCTD, and this interaction recruits αCTD, and hence the rest of RNA polymerase, to the target promoter (Busby and Ebright, 1994). Such activators can be considered as falling into two classes. Class I activators bind to a target site upstream of the promoter −35 region and interact with αCTD. One or both αCTDs are then recruited to the promoter DNA located between the activator and the −35 element. Class II activators bind to a target site that overlaps the promoter −35 region and, in many cases, make contact with domain 4 of the RNA polymerase σ subunit that is bound to the −35 element. Such activators may also contact the N-terminal domain of the α subunit (Busby and Ebright, 1999; Dove et al., 2003). In many instances, both αCTDs bind upstream of the class II activator (illustrated in Fig. 1A). In some cases, bound αCTD makes interactions with the activator that contribute to activation, while in other cases, αCTD binds at no unique location and makes little or no contribution. Study of other promoters that appear to be activated by a class II mechanism has revealed alternative locations for αCTD. Thus, at a promoter where multiple activators bind along one face of the DNA, αCTD appears to bind to the opposite face of the promoter that is not occupied by activator (illustrated in Fig. 1B; see Boucher et al., 2003). Yet another organization is found at the bacteriophage λ PRE promoter, where the activator, CII, binds on the opposite side of the DNA to the promoter −35 element. Thus, αCTD can bind adjacent to domain 4 of the RNA polymerase σ subunit (Kedzierska et al., 2004; illustrated in Fig. 1C).
In recent work, we sought to understand the regulation of the Escherichia coli melAB promoter (Belyaeva et al., 2000; Wade et al., 2000). This promoter, which controls expression of the melibiose operon, is co-dependent on activation by the cyclic AMP receptor protein (CRP) and MelR, a melibiose-triggered activator that is related to AraC (Wade et al., 2001). We identified four 18 bp DNA sites for MelR (Belyaeva et al., 2000; Howard et al., 2002) and showed that activation depends on the occupation of two of these sites, at position −62.5 (site 2) and position −42.5 (site 2′). We described two simplified melAB promoter derivatives, JK16 and JK19, that carry just MelR binding sites 2 and 2′ and show full MelR-dependent activation without CRP and without the upstream DNA sites for MelR at positions −120.5 (site 1′) and −100.5 (site 1) (Grainger et al., 2003). Site 2′ for MelR overlaps the melAB promoter −35 element. Thus, MelR appears to be a class II activator and, in the accompanying paper (Grainger et al., 2004), interactions with domain 4 of the RNA polymerase σ subunit are discussed.
Evidence for the importance of αCTD in E. coli mel operon expression first came from the observation that the phs mutation in rpoA (which results in the KE271 substitution in αCTD) downregulates the melAB promoter (Giffard and Booth, 1988; Zou et al., 1997). Subsequently, in vitro studies by Wade et al. (2001) showed that αCTD plays a role in MelR-dependent activation of the melAB promoter. Hence, in this work, we have focused on investigating this role, and we have exploited the simplified melAB promoter derivatives that carry just MelR binding site 2 and site 2′. During activation at these promoters, two bound MelR molecules occupy one face of the DNA from position −34 to position −71 and, thus, our first task was to identify binding locations for αCTD during transcription activation. To do this, we used preparations of RNA polymerase that had been reconstituted in vitro with α subunits that had been labelled at residue 302 (in αCTD) with a chemical nuclease, p-bromoacetamidobenzyl-EDTA-Fe (FeBABE). Footprinting with this nuclease was used to show that αCTD is located near position −52 and near position −72, i.e. just upstream of MelR bound at site 2′ and MelR bound at site 2, respectively, different from their positions with respect to other class II activators characterized to date. We present evidence to show that α interacts with MelR, and we have used alanine scanning to identify determinants in αCTD that are important for MelR-dependent activation of the melAB promoter.
Results and discussion
Binding of αCTD to the melAB promoter: hydroxyl radical footprinting
Transcription activation at the JK19 melAB promoter is dependent on MelR binding to two 18 bp target se-quences: site 2′, centred at position −42.5; and site 2, centred at position −62.5. The JK19 promoter was derived from the wild-type melAB promoter by improvements to MelR binding site 2′ that resulted in activation becoming independent of CRP and independent of MelR binding to sites located further upstream (Belyaeva et al., 2000). Grainger et al. (2003) used hydroxyl radical footprinting to study the binding of MelR to the JK19 melAB promoter. Binding results in the protection of five sets of bands, each set separated from its neighbour by 10–11 bp. This is the expected pattern from the binding of two MelR molecules to the adjacent site 2 and site 2′ sequences, with each subunit using its two helix–turn–helix (HTH) motifs to penetrate two neighbouring segments of the major groove on one face of the DNA target. Here, we have repeated the hydroxyl radical footprinting experiment with MelR, with and without the addition of RNA polymerase (Fig. 2). It is known that, during class II activation, αCTD of RNA polymerase binds to upstream promoter locations. Thus, we reasoned that comparison of the hydroxyl radical footprints of MelR with or without RNA polymerase might be informative about the location of αCTD during activation of the JK19 melAB promoter. Results illustrated in Fig. 2 confirm the footprints due to MelR reported by Grainger et al. (2003), and show clear protection downstream of site 2′, as expected, resulting from RNA polymerase. In the presence of RNA polymerase, supplementary protection is observed near position −51, between MelR binding sites 2 and 2′. Weak protection is also seen near position −85, just upstream of site 2 (this protection has been quantified by densitometry). Although these effects could be indirect, caused by DNA conformational changes, the likely explanation is that they result from αCTD binding to promoter DNA.
Binding of αCTD to the melAB promoter: studies with FeBABE-tagged RNA polymerase
To investigate further the location of αCTD during MelR-dependent activation of the JK19 melAB promoter, we exploited FeBABE, a chemical nuclease that can be covalently attached at specific locations in proteins. In previous work, we described the overexpression and purification of RNA polymerase α subunits containing a single cysteine residue at position 302 (in αCTD) that can be labelled with FeBABE (Lee et al., 2004). RNA polymerase could be reconstituted with this FeBABE-labelled Cys-302 α and bound at promoters. After triggering the nuclease activity of the FeBABE reagent, the location of αCTD can be deduced from the sites of DNA cleavage. Figure 3 shows the results of such an experiment to investigate the location of αCTD binding during activation of the JK19 melAB promoter by MelR. Three sites of DNA cleavage are observed. The strongest cleavage is located near position −51, between MelR binding site 2 and site 2′. Weaker cleavage also occurs upstream of MelR binding site 2, near position −75 and position −85. As these locations for αCTD are consistent with those suggested from hydroxyl radical footprinting, the likely interpretation is that one αCTD binds between MelR binding site 2 and site 2′ and the other binds upstream of site 2.
Open complex formation at the melAB promoter requires αCTD
The proximity of αCTD and MelR, when bound to the JK19 melAB promoter, suggests that they may interact during transcription activation. To investigate the role of αCTD, potassium permanganate footprinting was used to monitor open complex formation at the JK19 melAB promoter, using RNA polymerase reconstituted with full-length α subunits (WT RNA polymerase) or with truncated α subunits lacking αCTD (Δ235 RNA polymerase). Results illustrated in Fig. 4 show that, with WT RNA polymerase (lanes 2 and 3), open complex formation at the melAB promoter is dependent on MelR and that, as expected, bases from position −4 to position −11 become sensitive to permanganate. Interestingly, in both the absence and the presence of MelR, bases at positions +16 and +18 are permanganate sensitive: we attribute this to RNA polymerase binding to a weak downstream secondary promoter. Figure 4 (lane 4) shows that, with Δ235 RNA polymerase, MelR-dependent reactivity of bases from positions −4 to −11 is not observed. This shows that αCTD is required for MelR-dependent activation in this assay. Note that removal of αCTD in Δ235 RNA polymerase also reduces the activity of the downstream secondary promoter.
In a complementary experiment, purified full-length α subunits were added to the incubation with MelR and Δ235 RNA polymerase. The result in Fig. 4 (lane 5) shows that the inability of Δ235 RNAP to form a MelR-dependent open complex is reversed by the purified α protein. This argues that MelR and α interact to promote open complex formation at the JK19 melAB promoter. Note that the addition of α has no effect on the secondary downstream promoter.
Binding of MelR to the melAB promoter: effects of α
In the next set of experiments, we investigated the possible effects of α protein on the binding of MelR at site 2 and site 2′ of the JK19 melAB promoter. To do this, we exploited a preparation of purified MelR carrying the FeBABE reagent specifically attached at residue 269 (Grainger et al., 2003). We showed previously that this FeBABE-tagged MelR mediates cutting of JK19 promoter DNA at four adjacent sections of the minor groove, separated from each other by 10 bp. Two regions of cleavage, located near to positions −60 and −50, result from MelR binding to site 2, whereas the other two sets of DNA cleavage, located near positions −40 and −30, result from MelR binding to site 2′ (Grainger et al., 2003). Results illustrated in Fig. 5 show that the addition of purified α protein causes changes in the relative amounts of cleavage at the different locations. Thus, cutting near positions −30 and −40, resulting from MelR at site 2′, increases, whereas cutting near position −50, resulting from MelR binding at site 2, is decreased. This suggests that α protein stabilizes MelR binding at site 2′.
To investigate further the effects of α protein on the binding of MelR at the JK19 melAB promoter, we used electrophoretic mobility shift assays (EMSAs). Figure 6 illustrates an experiment in which increasing concentrations of purified MelR were added to 32P-labelled JK19 DNA in the presence or absence of purified α protein. As expected, two distinct MelR–DNA complexes, *1 and *2, form: the *1 complex results from the binding of a single MelR molecule, and the *2 complex results from two molecules of MelR binding (Howard et al., 2002). In the presence of α protein, both complexes form at a lower concentration of MelR, providing further evidence for interactions between α protein and MelR bound at the melAB promoter.
Identification of residues in αCTD required for activation of the melAB promoter
In the next set of experiments, we tried to identify αCTD residues that contribute to MelR-dependent activation of the melAB promoter using alanine scanning. Thus, derivatives of pHTf1α and pREIIα, which encode mutant α with a single alanine substitution at each position in αCTD, were transformed into WAM132 ΔmelR Δlac cells carrying pLG314, encoding melR, and pRW50, carrying different melAB promoter::lac fusions. The effect of each different alanine substitution was estimated after measurement of β-galactosidase expression in each transformant. Measurements were made with the JK19 promoter, the JK16 derivative (that is also CRP independent but has a lower affinity for MelR than JK19; Grainger et al., 2003) and the ‘wild-type’ KK43 melAB promoter.
Figure 7A shows a graphic representation of the data, focusing on locations where alanine substitution results in a decrease in expression of more than 20%. The results show that the effects of the different substitutions vary according to the promoter. Thus, at the JK19 promoter, only two substitutions, EA288 and LA289, cause a significant decrease. At the JK16 promoter, the EA273, VA287 and KA297 substitutions, but not EA288 and LA289, reduce transcription activation. The locations of these substitutions in αCTD are shown in Fig. 7B. It is apparent that E273, V287, E288 and L289 form a surface-exposed patch on αCTD, which is separate from K297 (part of the αCTD DNA-binding determinant). With the KK43 promoter, which carries the wild-type melAB promoter sequence, alanine substitutions at residues V264, S266, K271, V287 and K297 disrupt transcription activation. With the exception of K271, these residues are within or near the αCTD DNA-binding determinant or part of the surface that is required for activation at the JK19 and JK16 promoters. Interestingly, certain alanine substitutions lead to increases in MelR-dependent expression. We suppose that some of these substitutions create favourable MelR–αCTD interactions, while others remove clashes, but we cannot provide a full explanation for these effects.
Wade et al. (2001) showed that RNA polymerase reconstituted with α subunits lacking αCTD is defective in MelR-dependent activation of the melAB promoter, and this has been confirmed here by the assays for DNA opening shown in Fig. 4. Our aim in this work was to investigate the role of α at the melAB promoter and to establish whether it interacts with MelR. To do this, we used the simplified JK19 melAB promoter that is dependent on MelR alone. Our previous results had suggested that MelR functions as a class II activator and thus our first task was to determine the location of αCTD in ternary MelR–RNA polymerase–promoter complexes. Two independent pieces of experimental evidence, hydroxyl radical footprinting and the DNA cleavage patterns with RNA polymerase containing FeBABE-labelled Cys-302 α subunits, argue that the two αCTDs are located just upstream of MelR bound at site 2 and MelR bound at site 2′, as illustrated in Fig. 8. This represents a variation of the current models for class II activation (summarized in Fig. 1). However, this is consistent with previous thinking about class II activation that suggests that the two RNA polymerase αCTDs often occupy the first available upstream ‘slots’ on promoter DNA.
At many class II activator-dependent promoters, αCTD interacts with the activator, and this interaction contributes to promoter strength. The experiments illustrated in Figs 4, 5 and 6 argue for such an interaction and, in our system, we were able to monitor interactions between MelR and purified RNA polymerase α subunits in the absence of the rest of RNA polymerase. In the final part of this study, we exploited alanine scanning of αCTD to identify amino acid side-chains that play a role in MelR-dependent activation at the melAB promoter. This approach has been used identify αCTD residues important for transcription activation mediated by a number of different transcription factors (Savery et al., 1998; Holcroft and Egan, 2000a,b; Ruiz et al., 2001; Aiyar et al., 2002; Savery et al., 2002; Kedzierska et al., 2004). Using the JK19 melAB promoter derivative, we found only two alanine substitutions that substantially decrease MelR-dependent activation. These substitutions fall at E288 and L289. As these are surface-exposed residues that are far from the DNA-binding surface of αCTD, we conclude that they may identify the αCTD surface that contacts MelR. With the related JK16 promoter, we identified E273, V287 and K297 as the key residues. E273 and V287 are adjacent to E288 and L289 and, presumably, form part of the same contact surface, whereas K297 is a residue involved in αCTD binding to DNA. Taken together, these results argue that the αCTD surface containing residues E273, V287, E288 and L289, highlighted in red in Fig. 7B, forms the contact surface for MelR. Interestingly, a similar experiment with the wild-type melAB promoter carried on the KK43 promoter identified different residues as playing the most important roles (Fig. 7A). From this, we conclude that the relative role of specific residues within the target patch on αCTD must depend on the precise organization of the target promoter. Some of the differences can probably be attributed to alterations in DNA bending that affect the relative positioning of MelR and αCTD. Further studies will be needed to understand these differences at different melAB promoter derivatives.
Strains, plasmids and promoter fragments
Bacterial strains, plasmids and the melAB promoter fragments used in this work are listed in Table 1. Standard techniques for recombinant DNA manipulations were used throughout.
Table 1. . Bacterial strains, plasmids and promoter fragments.
For EMSA and standard footprinting experiments, MelR was overexpressed in BL21 (λDE3) cells, carrying the plasmids pLysS and pCM117-303, and purified as described by Michán et al. (1995). For the FeBABE experiments, we used a derivative of pCM117-303, encoding MelR with a single cysteine at position 269 of the protein. MelR used for FeBABE experiments was purified and conjugated with FeBABE according to the protocol of Grainger et al. (2003). Protein concentrations were determined by the protocol of Bradford (1976) using bovine serum albumen as a standard.
Purification of α subunits and reconstitution of RNA polymerase
RNAP α subunits carrying a hexahistidine tag between the first and second codons were overexpressed in the strain BL21(λDE3) using the plasmid pHTT7f1NHα exactly as in our previous work (Savery et al., 1998). Truncated α subunits, lacking residues 236–329, used to reconstitute Δ235 RNAP, were overexpressed using the plasmid pREII-NHα (1–235) in the strain XL1-blue and purified according to the protocol of Niu (1999). Other subunits of RNAP were overexpressed, purified and reconstituted with α to form active RNAP as described by Savery et al. (1998). RNA polymerase reconstituted with FeBABE-labelled Cys-302 α was prepared as described by Lee et al. (2004). Concentrations of RNA polymerase and purified subunits were determined by the Bradford (1976) method.
Preparation of DNA fragments
DNA fragments were derived from caesium chloride-purified preparations of plasmid pAA121 carrying the JK19 melAB promoter fragment. AatII–HindIII fragments were purified and labelled at the HindIII end with [γ-32P]-ATP and polynucleotide kinase. For all experiments, DNA fragments were used at a final concentration of 10 nM in buffer containing 60 mM Hepes, pH 8.0, 1.25 µM potassium glutamate and 40 µg ml−1 herring sperm DNA.
Electrophoretic mobility shift assays
DNA fragments were incubated with 4 µM RNAP α subunits or the appropriate volume of a storage buffer for 10 min at room temperature. MelR was then added to a final concentration of between 0.1 and 0.4 µM. After a further 10 min incubation, glycerol was added to the reaction to a final concentration of 6.5% (v/v), and samples were applied to a 7.5% polyacrylamide gel and run in a standard Tris-borate/EDTA electrophoresis buffer at 100 V. After electrophoresis, gels were dried and processed using a Phosphorimager (Molecular Dynamics).
Potassium permanganate footprinting
DNA fragments were incubated with 0.75 µM MelR for 10 min at room temperature. RNA polymerase α protein was then added to a concentration of 4 µM and incubated for a further 10 min (α storage buffer was used in experiments in which no free a protein was added). Finally, RNA polymerase was added to the reaction mixture that was incubated for 20 min at 37°C. Reaction mixtures were then treated with potassium permanganate and piperidine exactly as described by (Savery et al., 1996). Modified DNA was extracted with phenol–chloroform and precipitated with ethanol before analysis on 6% polyacrylamide gels containing 6 M urea. Gels were calibrated with Maxam–Gilbert G+A sequencing ladders and processed and scanned using a Phosphorimager (Molecular Dynamics)
Hydroxyl radical and FeBABE footprinting protocols
FeBABE footprinting experiments using RNA polymerase reconstituted with FeBABE-labelled Cys-302 α subunits were performed as described by Lee et al. (2004). FeBABE footprinting experiments using FeBABE-labelled Cys-269 MelR were performed as described by Grainger et al. (2003). Hydroxyl radical footprinting was done according to the protocol of Savery et al. (1996). MelR was used at a final concentration of 0.75 µM, RNA polymerase was used at 0.2 µM and α protein was used at 4 µM. For experiments without added α protein, the appropriate volume of a storage buffer was added to reactions.
Measurement of promoter activities in vivo
DNA fragments containing the melAB promoter were cloned into pRW50, a low-copy-number lac expression vector, to generate pmelAB::lac fusions. β-Galactosidase levels in cells carrying these recombinants were measured by the Miller (1972) method. Activities shown are the average of at least three independent experiments, and error bars show one standard deviation either side of the mean. Cells were grown in media either with or without melibiose exactly as in our previous work (Webster et al., 1987): data shown are those obtained from cultures with melibiose, unless stated otherwise. Assays were performed in a ΔmelR Δlac (WAM132) background carrying different pREIIα or pHTf1α derivatives, encoding wild-type and mutant rpoA, and pLG314 encoding wild-type MelR.
This work was supported by the UK BBSRC with a project grant to S.J.W.B. and E.I.H., and by the UK MRC with a PhD studentship award to D.C.G. We are grateful to Rick Gourse, Wilma Ross and Nigel Savery for many helpful discussions.