Role of Activating Region 1 of Escherichia coli FNR Protein in Transcription Activation at Class II Promoters*

FNR is an Escherichia coli transcription factor that activates gene expression in response to anaerobiosis at a large number of promoters by making direct contacts with RNA polymerase. At class II FNR-dependent promoters, where the DNA site for FNR overlaps the 2 35 element, activating region 1 of FNR is proposed to inter-act with the C-terminal domain of the RNA polymerase a -subunit. Using a model class II FNR-dependent promoter, FF( 2 41.5) , we have performed in vivo and in vitro experiments to investigate the role of this interaction. Our results show that FNR, carrying substitutions in activating region 1, is compromised in its ability to promote open complex formation and thus to activate transcription. Abortive initiation assays were used to assess the contribution of activating region 1 of FNR to open complex formation. A new method for the purification of the FNR protein is also described.

Many bacterial gene regulatory proteins activate transcription by making direct contact with RNA polymerase holoenzyme (RNAP). 1 A large number of these activators contact RNAP via the C-terminal domain of the ␣-subunit (␣CTD) (1). One of the best studied examples of this type of activator is the Escherichia coli cAMP receptor protein, CRP (reviewed in Refs. 2 and 3). In response to glucose starvation, CRP is triggered by cAMP and activates transcription initiation at more than 100 different promoters. In its active form, CRP binds as a dimer to specific DNA sequences found at target promoters; the consensus site is a 22-base pair sequence that is organized as an 11-base pair inverted repeat. At CRP-dependent promoters, CRP activates transcription by making several direct contacts with RNAP. One of these contacts involves the interaction of a single surface-exposed ␤-turn of CRP (residues 156 -164) with ␣CTD. This region in CRP is known as activating region 1 (AR1). The role of AR1 of CRP is to enhance open complex formation at target promoters simply by increasing the initial binding of RNAP (reviewed in Ref. 2).
The FNR protein is another global activator of gene expression in E. coli, which regulates transcription initiation in re-sponse to oxygen starvation (4,5). FNR belongs to the same family of transcription factors as CRP, and the two proteins have related amino acid sequences (6). Although the high resolution structure of the FNR protein has not been determined, several crystallographic structures for CRP have been solved (7)(8)(9) and sequence alignments suggest that FNR and CRP have similar three-dimensional structures (4,6). Like CRP, FNR regulates transcription as a dimer and recognizes a 22base pair binding sequence at target promoters. However, dimerization of FNR is triggered by the anaerobic acquisition of a [4Fe-4S] 2ϩ center in each FNR subunit (10 -12). By analogy to CRP, during transcription initiation at FNR-dependent promoters it is proposed that FNR interacts with the ␣CTD of RNAP. Genetic analysis suggests that the amino acid side chains involved in this interaction are located in three adjacent surface-exposed loops, residues 71-75, 116 -121, and 184 -192 (13). These loops form an extended surface of FNR, also known as AR1, which is much larger than AR1 of CRP (although AR1 of FNR and CRP do overlap, the 184 -192 loop of FNR is equivalent to the ␤-turn containing residues 156 -164 in CRP).
The key residues in AR1 of FNR, which are likely to provide the crucial side chains for the FNR-␣CTD interaction, were identified as Thr 118 and Ser 187 (alanine substitutions at these positions reduce transcription activation by FNR) (13).
Most naturally occurring FNR-dependent promoters contain a DNA site for FNR that overlaps the Ϫ35 element (promoters organized in this way are known as class II promoters) (5). Previous studies with class II FNR-dependent promoters showed that AR1 of FNR is functional on the upstream subunit of the FNR dimer, and thus it is this subunit that is thought to contact ␣CTD (14). The aim of this work was to investigate the role of AR1 of FNR during transcription activation using in vitro approaches with the model class II FNR-dependent promoter, FF(-41. 5

EXPERIMENTAL PROCEDURES
Plasmid Construction--pFNR T118A/S187A was constructed by site-directed mutagenesis using PCR with pFNR T118A as template DNA (13). pFNR derivatives encoding FNR*, FNR* T118A, FNR* S187A, and FNR* T118A/S187A were also constructed by PCR using pFNR, pFNR T118A, pFNR S187A, and pFNR T118A/S187A, respectively, as templates. PCR products from these reactions were cut with BamHI and HindIII, and the resulting fragments were cloned into purified BamHI-HindIII vector from pFNR. The NcoI and BglII restriction sites, which were used to clone the different fnr derivatives into the pQE60 C-terminal His tag overexpression vector (supplied by QIA-GEN), were created by PCR. The resulting DNA was cut with NcoI and BglII, and fragments were cloned into the purified NcoI-BglII vector from pQE60 so that the fnr gene and the C-terminal His tag were in frame.
Overexpression and Purification of FNR* Derivatives-As a host background for the overexpression of the FNR* derivatives, the strain M15 (derived from E. coli K12; supplied by QIAGEN) carrying the plasmid pREP4 (derived from pACYC and encoding constitutively expressed LacI; supplied by QIAGEN) was used. Cells were transformed with pQE60 derivatives encoding different C-terminally His-tagged FNR* proteins, and transformants were grown at 37°C in 100 ml of L-broth with appropriate antibiotics until cultures reached an A 600 of 0.5-0.6. Overexpression of the His-tagged proteins was then induced by the addition of 0.1 M isopropyl-1-thio-␤-D-galactopyranoside for 1 h. Cells were harvested, and pellets were sonicated in 10 ml of lysis buffer at 4°C (1 mg/ml lysozyme, 50 mM NaH 2 PO 4 /Na 2 HPO 4 , pH 8.0, 0.75 M NaNO 3 , 10 mM imidazole, 10 mM benzamidine). Sonicates were centrifuged at 10,000 ϫ g, and the amount of each His-tagged FNR* derivative was estimated by SDS-polyacrylamide gel electrophoresis. Supernatants were applied to nickel-nitrilotriacetic acid-agarose (supplied by QIAGEN) columns at 4°C so that the binding capacity of the agarose (5-10 mg/ml) was exceeded (typical column volumes were 0.75-1.0 ml). Columns were then washed with 50 column volumes of wash buffer (50 mM NaH 2 PO 4 /Na 2 HPO 4 , pH 8.0, 0.75 M NaNO 3 , 20 mM imidazole), and FNR* derivatives were eluted using elution buffer (50 mM NaH 2 PO 4 / Na 2 HPO 4 , pH 8.0, 0.75 M NaNO 3 , 250 mM imidazole). Proteins were stored at 4°C in elution buffer where they remained stable for up to 6 months. Iron-sulfur clusters were assembled in anaerobic samples of purified FNR* proteins as described previously (20). The [4Fe-4S] 2ϩ contents of the reconstituted proteins were monitored by scanning the reconstitution reactions between 240 and 900 nm. The spectra were typical of those obtained with wild-type FNR. When reconstitutions were judged to be complete, the absorbance at 420 nm was measured, and the [4Fe-4S] cluster content was calculated using E 420 ϭ 13,300 M Ϫ1 cm Ϫ1 (20). As the His-tagged FNR* preparations were found to be fully functional (see "Results" below), the hexa-His tag was not removed from the FNR* preparations used in this work.
In Vitro Transcription Assays-To measure FNR-dependent transcription initiation in vitro we used the pSR plasmid described by Kolb et al. (21). The test promoter, FF(Ϫ41.5), was cloned into this plasmid upstream of the oop terminator, so that transcripts initiating at FF(Ϫ41.5) would be a discrete length. The protocol used was based on that described in (22): binding reactions (12 l of final volume) contained 5 nM FF(Ϫ41.5)/pSR, 25 nM RNAP (supplied by Amersham Pharmacia Biotech) and 500 nM of each FNR derivative. Labeled RNA products were separated on a 6% denaturing polyacrylamide gel and quantified using a Molecular Dynamics PhosphorImager and the software ImageQuant, v3.3.
DNase I and Potassium Permanganate Footprinting Experiments-Footprinting studies were performed on 32 P-end-labeled PstI-HindIII fragments containing the FF(Ϫ41.5) promoter, using the protocols described in Ref. 23. Each reaction contained a final concentration of 4 nM template DNA, 1.75 M FNR* or FNR* derivative, and 250 -500 nM RNAP (supplied by Amersham Pharmacia Biotech). The binding buffer used in these reactions contained 20 mM Hepes, pH 8.0, 5 mM MgCl 2 , 50 mM potassium glutamate, 1 mM dithiothreitol, 30 g/ml herring sperm DNA, and 500 g/ml bovine serum albumin. The RNAP concentration was lowered to 100 -350 nM in the potassium permanganate footprints because of the sensitivity of this assay. Footprinting gels were calibrated with Maxam-Gilbert "AϩG" sequencing reactions of the labeled fragments and quantified using a Molecular Dynamics PhosphorImager and the software ImageQuant, v3.3.
Abortive Initiation Assays-Template DNA was prepared by PCR amplification of the FF(Ϫ41.5) promoter. Binding reactions contained DNA, purified FNR* or mutant derivatives, ApU, and [␣-32 P]UTP in 15 l of binding buffer (5% (w/v) glycerol, 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM MgCl 2 , 0.1 mM EDTA, 1 mM dithiothreitol, 50 g/ml bovine serum albumin). After preincubation at 37°C for 10 min, reactions were started by adding 10 l of RNAP in binding buffer (this gave final reaction conditions of 2 nM template DNA, 350 nM FNR* derivative, 50 -150 nM RNAP, 0.5 mM ApU, 50 M UTP, and 0.75 Ci [␣-32 P]UTP). At regular time intervals, 2-l samples were removed from the reactions and terminated by adding 3 l of stop buffer (20 mM EDTA, 80% (v/v) deionized formamide, 0.1% (w/v) xylene cyanol, 0.1% (w/v) bromphenol blue). The reaction product, ApUpU, and unincorporated UTP were separated on 20% denaturing polyacrylamide gels and quantified using a Molecular Dynamics PhosphorImager and the software Image-Quant, v3.3 (as described in Ref. 24). The kinetic constants for transcription initiation at FF(Ϫ41.5) were calculated by measuring the rate of ApUpU production over a range of RNAP concentrations and by plotting the lag time () of these reactions against the reciprocal of RNAP concentration.
Heparin Challenge Experiments-Template DNA was prepared by PCR amplification of the FF(Ϫ41.5) promoter. Binding reactions contained DNA, purified FNR* or mutant derivatives, and RNAP in 20 l of binding buffer (5% (w/v) glycerol, 20 mM Tris-HCl, pH 8.0, 100 mM NaCl . After incubation at 37°C, 2 l of each reaction was removed and added to 3 l of stop buffer. The reaction product, ApUpU, and unincorporated UTP were separated on 20% denaturing polyacrylamide gels and quantified using a Molecular Dynamics PhosphorImager and the software ImageQuant, v3.3 (as described in Ref. 24). The fraction of the open complex remaining was calculated from the amount of ApUpU produced. The decay rate of this fraction was calculated for each heparin concentration, and the backward rate constants for isomerization (k Ϫ2 ) of FF (-41.5) in the presence of FNR* or FNR* T118A/S187A were determined by extrapolating these data to a zero heparin concentration.

RESULTS
In Vivo Transcription Assays-The starting point of this work was the pFNR plasmid that encodes FNR. A derivative of pFNR, pFNR*, encoding FNR with the D154A substitution, known to confer FNR activity in the presence of oxygen (15), was constructed. The aim of this work was to investigate the role of AR1 in transcription activation by FNR at class II promoters. Thus, three derivatives of pFNR* were constructed that encoded alanine substitutions at either or both of the key residues in AR1: pFNR* T118A, pFNR* S187A, and pFNR* T118A/S187A. To check that FNR* was able to function aerobically and to investigate the effects of the different AR1 substitutions when combined with D154A, transcription activation was measured in vivo at the FF(Ϫ41.5) promoter. The FF(Ϫ41.5) promoter is an artificial class II FNR-dependent promoter that contains a consensus DNA site for FNR centered between 41 and 42 base pairs upstream of the melR transcript start site (i.e. at position Ϫ41.5). The ⌬lac ⌬fnr strain, JRG1728, carrying the FF(Ϫ41.5)::lacZ fusion in pRW50 (14), was transformed with the four different pFNR* derivatives. Levels of transcription from FF(Ϫ41.5) were determined by measuring ␤-galactosidase expression in cells that had been grown aerobically or anaerobically. The data in Table I show that, in the absence of FNR*, under both aerobic and anaerobic conditions, transcription from the FF(Ϫ41.5) promoter is very low. Expression from FF(Ϫ41.5) is clearly activated by FNR* under both aerobic and anaerobic conditions, but FNR* variants carrying either or both of the alanine substitutions in AR1 are compromised in their ability to activate transcription. Thus, FNR* is able to activate transcription under aerobic conditions and activation is disrupted by the different AR1 substitutions. These data suggest that these FNR derivatives could be used in in vitro studies at the FF(Ϫ41.5) promoter.
Purification and Properties of FNR* and FNR* Carrying AR1 Substitutions-To investigate the effects of substitutions in AR1 of FNR in vitro, His-tagged FNR*, FNR* T118A, FNR* S187A, and FNR* T118A/S187A were purified from cell lysates using nickel affinity chromatography. Typically, culture volumes of 100 ml yielded 300 -400 mg of soluble protein, of which 20 -25% was His-tagged FNR* protein. Affinity chromatography yielded 2-8 mg of the different His-tagged FNR* derivatives, and the protein preparations were estimated to be 90% pure by SDS-polyacrylamide gel electrophoresis. All of the buffers used throughout this procedure contained 0.75 M NaNO 3 , which was found to improve the solubility and stability of the FNR* proteins. After anaerobic reconstitution by the method of Green et al. (20), His-tagged FNR* and His-tagged FNR* T118A/S187A had optical spectra indistinguishable from those of unaltered FNR. Assuming that all the absorbance at 420 nm was because of the [4Fe-4S] form of FNR, the FNR molar extinction co-efficient (E 420 ϭ 13,300 M Ϫ1 cm Ϫ1 ) was used to calculate that our FNR preparations contained 0.95 clusters/ FNR subunit. Exposure of the reconstituted proteins to air caused the loss of the broad absorbance around 420 nm, indicating that cluster disassembly had occurred. Therefore, the estimates of [4Fe-4S] content, the typical shape of the spectra, and the oxygen sensitivity of the clusters provide good evidence to suggest that neither the His tag nor the amino acid replacements in AR1 grossly distort the protein structure. Thus, these studies indicate that the impairment of transcription activation associated with the amino acid substitutions in AR1, observed both aerobically and anaerobically in vivo (Table I), is because of a defective AR1 rather than the failure to acquire a [4Fe-4S] 2ϩ center.
In Vitro Transcription Assays-In vitro transcription assays were used to investigate the effects of different substitutions in AR1 of FNR. Fig. 1 shows a typical result using the FF(Ϫ41.5) promoter cloned upstream of the oop terminator in a supercoiled plasmid, in the presence of purified RNAP and the different purified FNR* variants. Transcription initiation from FF(Ϫ41.5) is clearly activated by FNR*. This activation is reduced by the different alanine substitutions in AR1, confirming the effects observed in vivo, and showing that substitutions in AR1 compromise the ability of FNR* to activate transcription at the FF(Ϫ41.5) promoter.
DNase I and Potassium Permanganate Footprinting Experiments-To study open complex formation in the presence of FNR* or FNR* carrying substitutions in AR1, we used DNase I and potassium permanganate footprinting. Saturating amounts of FNR* or FNR* T118A/S187A (350 nM in both cases) and a range of RNAP concentrations were used to form open complexes at the FF(Ϫ41.5) promoter. The DNase I footprints (Fig. 2) show that, when either FNR* or FNR* T118A/S187A is incubated with the promoter fragment alone, a region centered at position Ϫ41.5, relative to the transcription start point, is protected (lanes c and k). Thus both FNR* derivatives recognize and bind to the FNR target site. With FNR*, increasing concentrations of RNAP result in increasing protection of the promoter region both upstream and downstream of the FNR binding site (lanes d-i). Based on footprinting studies of CRP The autoradiograph shows RNA transcripts generated from the FF(Ϫ41.5) promoter carried by the plasmid pSR. Incubations contained RNAP and FNR* or different mutant derivatives, as indicated. Transcripts initiating at FF(Ϫ41.5) run to a strong terminator and give bands that are labeled accordingly. The RNAI transcript, encoded by the pSR vector, serves as an internal control, and the end-labeled oligonucleotide provides a loading control. Transcripts initiating from FF(Ϫ41.5) were quantified and normalized using the control bands. The relative band intensities are: 100% for FNR*, 28% for FNR* S187A, 30%for FNR* T118A, and 13% for FNR* T118A/S187A. FF(؊41.5). The autoradiograph shows a sequencing gel run to analyze products after DNase I cleavage of complexes formed at FF(Ϫ41.5) with FNR* (lanes c-i) or FNR* T118A/S187A (lanes k-q) and increasing concentrations of RNAP (ranging from 250 to 500 nM, at 50 nM intervals). Lane j shows the footprint obtained with RNAP alone (500 nM). A Maxam-Gilbert AϩG sequencing reaction (lane a) was used to calibrate the gel; ϩ1 denotes the FF(Ϫ41.5) transcript start point. The vertical bars indicate regions of protection by FNR* derivatives and RNAP. and RNAP at class II CRP-dependent promoters, the upstream protection can be attributed to the ␣CTD of RNAP (25,26). Footprints with FNR* T118A/S187A also show increasing protection with increasing RNAP concentrations (lanes l-q), although significantly less protection is seen in comparison to the footprints with FNR*. Interestingly, the pattern of protection of ternary complexes containing either FNR* or FNR* T118A/ S187A is the same. This is in contrast to the situation observed at class II CRP-dependent promoters, where substitutions in AR1 of CRP induce changes in the DNase I footprint in the region of the CRP-␣CTD interface (26). Strikingly, in the experiment described here, no protection is seen with RNAP alone, even at 500 nM (lane j), showing that RNAP alone cannot form stable complexes at the FF(Ϫ41.5) promoter.

FIG. 2. DNase I footprint analyses of complexes formed at
Potassium permanganate footprints show the appearance of bands because of unpaired thymine bases that result when the DNA duplex is unwound near the transcript start point during open complex formation. The results in Fig. 3A show that, at FF(Ϫ41.5), bands because of oxidation of these thymine bases appear at positions ϩ1, Ϫ2, Ϫ8, Ϫ9, and Ϫ11, with both FNR* (lanes c-h) and FNR* T118A/S187A (lanes j-o), as RNAP is added. However, as the RNAP concentration is increased, the intensity of the bands in lanes containing FNR* T118A/S187A is significantly lower than in the lanes containing FNR* (see Fig. 3B). Note that thymine-sensitive bands are not seen with RNAP alone (lane i), implying that promoter opening is completely FNR-dependent.
Abortive Initiation Assays-The in vitro footprint experi- The lag time observed before linear production of ApUpU is plotted against the reciprocal of RNAP concentration. The plot compares FNR* T118A/S187A with FNR*. Each data point represents the average of five independent assays, and the error bars show one standard deviation either side of the mean. Values for k 2 and K B k 2 , shown below the graph, were calculated from the intercepts and slopes of the plots, respectively. The tight coupling of FF(Ϫ41.5) activity to FNR* precludes the accurate measurement of k 2 and K B in the absence of FNR*. To investigate further the consequences of alterations in AR1, abortive initiation assays were used to attempt to measure the binding constant for RNAP (K B ) and the forward rate constant for isomerization from the closed to open complex (k 2 ) during transcription activation by FNR* or FNR* T118A/S187A at FF(Ϫ41.5). The rate of ApUpU production at FF(Ϫ41.5) was measured in the presence of FNR* or FNR* T118A/S187A, and the resulting plots are shown in Fig. 4. Although our data are subject to large errors, we can conclude that the alanine substitutions in AR1 of FNR* cause a reduction in k 2 . However, the errors involved in calculating K B are so large that no reliable comparison of K B values for FNR* and FNR* T118A/S187A can be made.
Heparin  Fig. 5 show that the values of k Ϫ2 are negligible for both FNR* and FNR* T118A/S187A (more than 80-fold lower than k 2 ). This shows that open complexes formed in the presence of both FNR* and FNR* T118A/S187A are stable. This rules out the possibility that the substitutions in AR1 of FNR affect the stability of the open complex and validates the conclusions drawn from the plots (Fig. 4). DISCUSSION Many bacterial transcription factors activate transcription by making direct contact with the ␣CTD of RNAP. For example, at CRP-dependent promoters the AR1-␣CTD interaction helps to recruit RNAP to the promoter DNA (2). Previous studies have suggested that FNR also functions by interacting with ␣CTD and have identified Thr 118 and Ser 187 as the crucial side chains of the AR1 equivalent in FNR (13,14). In this work, we have used in vitro studies to show that FNR carrying the T118A and S187A substitutions is indeed defective in transcription activation and is less able to promote the formation of open complexes. Using abortive initiation assays, we have assessed the role of AR1 of FNR. Although the large errors prevent an accurate estimation of k 2 and K B , the results suggest that AR1 of FNR plays a role in the transition from a closed to an open complex (at least at the FF(Ϫ41.5) promoter). This is in contrast to the situation with CRP, where AR1 has been reported to function solely in the initial recruitment of RNAP to promoter DNA (2). Thus, whereas both CRP and FNR function by interacting directly with ␣CTD, the consequences of these interactions appear to differ. This hypothesis is supported by the observation that FNR fails to recruit purified ␣ in electromobility shift assays (data not shown), unlike CRP, which binds ␣ cooperatively (28). Interestingly, the surface of FNR that is responsible for contacting ␣CTD is much larger than the equivalent surface of CRP (2,13). Further studies are needed to determine the individual contributions of the subregions of AR1 to the process of transcription activation.