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© 1997 Oxford University Press 4028-4034

Transcription activation at Class I FNR-dependent promoters: identification of the activating surface of FNR and the corresponding contact site in the C-terminal domain of the RNA polymerase [alpha] subunit

Transcription activation at Class I FNR-dependent promoters: identification of the activating surface of FNR and the corresponding contact site in the C-terminal domain of the RNA polymerase [alpha] subunit Stephen M. Williams, Nigel J. Savery, Stephen J. W. Busby* and Helen J. Wing

School of Biochemistry, University of Birmingham, Birmingham B15 2TT, UK

Received July 7, 1997; Revised and Accepted August 26, 1997

ABSTRACT

A library of random mutations in the Escherichia coli fnr gene has been screened to identify positive control mutants of FNR that are defective in transcription activation at Class I promoters. Single amino acid substitutions at D43, R72, S73, T118, M120, F181, F186, S187 and F191 identify a surface of FNR that is essential for activation which, presumably, makes contact with the C-terminal domain of the RNA polymerase [alpha] subunit. This surface is larger than the corresponding activating surface of the related transcription activator, CRP. To identify the contact surface in the C-terminal domain of the RNA polymerase [alpha] subunit, a library of mutations in the rpoA gene was screened for [alpha] mutants that interfered with transcription activation at Class I FNR-dependent promoters. Activation was reduced by deletions of the [alpha] C-terminal domain, by substitutions known to affect DNA binding by [alpha], by substitutions at E261 and by substitutions at L300, E302, D305, A308, G315 and R317 that appear to identify contact surfaces of [alpha] that are likely to make contact with FNR at Class I promoters. Again, this surface differs from the surface used by CRP at Class I CRP-dependent promoters.

INTRODUCTION

The Escherichia coli FNR protein is a global activator of transcription initiation which regulates transcription from a large number of promoters in response to oxygen starvation. FNR is related to another global regulator, the cyclic AMP receptor protein (CRP). FNR and CRP are believed to have similar structures and to have evolved from a common origin (reviewed in 1 ,2 ). Binding sites for both FNR and CRP span 22 bp, accommodating dimers of each activator. A striking feature of both FNR- and CRP-dependent promoters is that the location of the DNA site for the activator can vary from one promoter to another. Studies with semi-synthetic promoters in which a consensus DNA site for either FNR or CRP was positioned at different distances upstream of the same promoter elements showed that FNR or CRP dimers could activate transcription when they were centred near positions 41, 61, 71, 81 or 91 bp upstream of the transcript start point (3 -5 ).

At promoters where the DNA site for FNR or CRP is centred around 41 bp upstream of the transcription start (known as Class II promoters), FNR and CRP function by making multiple interactions with different parts of the RNA polymerase holoenzyme (RNAP) (reviewed in 6 ; see also 5 ). In contrast, at promoters where the DNA site for FNR or CRP is centred further upstream (known as Class I promoters), transcription activation is dependent solely on interactions with the C-terminal domain of the RNAP [alpha] subunit ([alpha]CTD) (reviewed in 7 ,8 ; see also 5 ). At Class I CRP-dependent promoters a surface-exposed loop on the downstream subunit of the CRP dimer (amino acids 156-164, known as activating region 1) interacts with a contact site in [alpha]CTD, resulting in recruitment of [alpha]CTD to promoter DNA and an increase in RNAP binding (reviewed in 7 ,8 ). Thus, transcription activation at Class I CRP-dependent promoters is suppressed by single amino acid substitutions in activating region 1 of CRP (9 ,10 ) and by deletions and single amino acid substitutions in [alpha]CTD (11 -14 ).

In contrast, far less is known about interactions between FNR and [alpha]CTD at Class I FNR-dependent promoters. A single positive control FNR mutant defective at Class I promoters has been identified (15 ). This mutant carries the single amino acid substitution SF73 and, based on its properties, Wing et al. (5 ) concluded that FNR must contain an activating surface that was equivalent to activating region 1 of CRP. Using the technique of `oriented heterodimers', this activating region was shown to be functional in the downstream subunit of the FNR dimer at Class I FNR-dependent promoters (5 ). In the first part of this work we generated a random library of mutations in the fnr gene and screened for FNR mutants that were defective in activation at Class I FNR-dependent promoters. This allowed us to identify the activating surface of FNR (including S73) that is functional at these promoters. In the second part we generated a random library of mutations in the segment of the rpoA gene encoding [alpha]CTD and screened for mutants that interfere with activation at Class I FNR-dependent promoters. The resulting mutants fall into a number of classes that confirm the role of [alpha]CTD in transcription activation and suggest a likely contact site for the activating region of FNR.

Table 1 Promoters and plasmids used in this work
Promoters (all cloned on fragments with EcoRI site upstream and HindIII site downstream of the transcription start)
FF(-71.5)

 Semi-synthetic FNR-dependent promoter with FNR binding site centred at position -71.5 upstream of the melR transcription start (5)
YF(-71.5)

 Derivative of FF(-71.5) with upstream half of FNR binding site, 5'-AAATTTGATGT-3' (designated F) changed to 5'-AAATTTAATGT-3' (designated Y) (5)
FY(-71.5) Derivative of FF(-71.5) with downstream F sequence replaced with Y sequence (5)
FF (-61.5)

 Semi-synthetic FNR-dependent promoter with FNR binding site centred at position -61.5 upstream of the melR transcription start (5)
pndh E.coli ndh promoter, which is repressed by FNR (15,17)
Plasmids
pRW50

 Broad host range lac expression vector for cloning of different promoters on EcoRI-HindIII fragments: encodes resistance to 35 µg/ml tetracycline (18)
pAA121

 General cloning vector for EcoRI-HindIII fragments derived from pBR322: encodes resistance to 80 µg/ml ampicillin (19)
pFNR

 Plasmid carrying fnr gene (and mutant derivatives) cloned in pBR322: encodes resistance to 80 µg/ml ampicillin (15)
pHW1

 Plasmid carrying fnr gene (and mutant derivatives) cloned in pLG339: encodes resistance to 25 µg/ml kanamycin (5,15)
pLAW2

 Plasmid carrying rpoA gene (and mutant derivatives) cloned in pBR322: encodes resistance to 80 µg/ml ampicillin (12)

MATERIALS AND METHODS

The [Delta]lac strains E.coli M182 fnr+ and JRG1728 [Delta]fnr were used throughout this work, as before (5 ,15 ). The plasmids and promoters used in this work are listed in Table 1 . All the promoters used were cloned on EcoRI-HindIII fragments and were shuttled between pAA121 (for manipulation) and pRW50 (for lac fusions and assays). By convention, promoter sequences are numbered with the transcript start as +1, with upstream and downstream sequences denoted by - and + prefixes respectively. Standard recombinant DNA, site-directed mutagenesis and sequencing technologies were used as in our previous work (5 ,15 ).

A library of random mutations throughout the fnr gene cloned in pFNR was created using error-prone PCR, exploiting the exact protocol described by Bell and Busby (15 ). From this library, positive control FNR mutants that were defective in FNR-dependent activation at the FF(-71.5) promoter were selected, using the protocol used by Bell and Busby (15 ) to obtain similar mutants at FF(-41.5). Briefly, M182 [Delta]lac fnr+ cells carrying pRW50 containing the FF(-71.5) promoter, encoding a FF(-71.5)::lac fusion, were transformed using electroporation with the pFNR mutant library. Transformants were plated on MacConkey lactose plates containing ampicillin and tetracycline, Lac- candidates were picked and purified and the pFNR derivative was extracted. We checked the ability of each putative mutant FNR to repress the ndh promoter and assayed activation at both FF(-61.5) and FF(-71.5). To do this, the mutant pFNR derivative was transformed into JRG1728 [Delta]lac [Delta]fnr cells carrying pRW50 into which the ndh promoter or FF(-61.5) or FF(-71.5) had been cloned. [beta]-Galactosidase activities of transformants grown anaerobically in L-broth supplemented with 0.4% glucose, ampicillin and tetracycline were measured (full details are given in 15 ). In this study we retained only those FNR mutants that, like wild-type FNR, fully repressed expression from the ndh promoter. The sequence of these mutants was deduced (Table 2 ) and the defect in transcription activation at FF(-61.5) and FF(-71.5) was quantified (Table 3 ). Derivatives of pFNR encoding FNR with single alanine substitutions at particular positions were made by PCR (16 ). In all cases the base sequence of the entire fnr gene was confirmed (secondary substitutions are noted in Table 2 ). Oriented heterodimer experiments (Table 5 ) were performed exactly as described by Bell and Busby (15 ) and Wing et al. (5 ).

Mutagenesis of rpoA encoded by pLAW2 was performed using protocols derived from Zou et al. (12 ). This plasmid carries a unique HindIII site adjacent to codon 231 of rpoA and a unique BamHI downstream of the rpoA stop codon. Using primers flanking the HindIII and BamHI sites the segment of rpoA corresponding to the C-terminal domain (from codon 231) was synthesized using error-prone PCR as above. After restriction with HindIII and BamHI, the product was cloned into pLAW2 to generate a library of random mutations in the segment of pLAW2 encoding [alpha]CTD. M182 [Delta]lac fnr+ cells carrying pRW50 containing the FF(-71.5) promoter, encoding a FF(-71.5)::lac fusion, were transformed using electroporation with the pLAW2 mutant library. Transformants were plated on MacConkey lactose plates containing ampicillin and tetracycline and Lac- candidates were picked and purified. The pLAW2 derivatives were extracted, mutant rpoA sequences were determined and the defects in transcription activation at FF(-71.5) were quantified (Table 6 ).

Table 2 . Sequences of 22 fnr-positive control (p.c.) mutants
Amino acid substitution Codon substitution No. of independent isolatesa Repression at pndhb (%)
DG43 GAT -> GGT 1 100
RH72 CGC -> CAC 1 88
SF73 TCC -> TTC 1 97
TA118 ACC -> GCC 1 98
TP118 ACC -> CCC 1c 100
MI120 ATG -> ATA 2 93
MR120 ATG -> AGG 1d 100
MT120 ATG -> ACG 2 100
MV120 ATG -> GTG 3e 94
FL181 TTT -> CTT 2 101
FS186 TTC -> TCC 2 105
SP187 TCC -> CCC 2f 95
FL191 TTC -> CTC 3 99
pFNR derivatives encoding FNR carrying the listed substitutions were isolated after mutagenesis of pFNR as described in the text. In each case the entire fnr base sequence was determined. JRG1728 cells carrying pRW50 containing the ndh promoter were transformed with different pFNR derivatives and [beta]-galactosidase expression in transformants was measured and compared with a control pFNR plasmid from which the fnr gene had been removed.
aIndependent isolates are defined as substitutions that occurred in different PCR reactions.
bRepression was measured in vivo using a pndh::lac fusion. Values of repression are expressed as percentages of repression achieved with wild-type FNR.
cThis isolate contained a second amino acid substitution MV223 (ATG -> GUG). Data for repression is for FNR carrying the single substitution mutant TP118, constructed by subcloning.
dThis isolate contained a second amino acid substitution YC230 (TAC -> TGC). Data for repression is for FNR carrying the single substitution MR120, constructed by subcloning.
eOne isolate contained two further amino acid substitution: FL191 (TTC -> CTC), KR220 (AAA -> AGA).
fOne isolate contained a second amino acid substitution: FY112 (TTC -> TAC).

Table 3 Transcription activation by FNR derivatives carrying different p.c. substitutions
Amino acid substitution Activation at FF(-71.5) (%) Activation at FF(-61.5) (%)
Wild-type FNR 100 100
DG43 42 32
RH72 20 13
SF73 5 6
TA118 15 19
TP118 18 16
MI120 8 9
MR120 3 1
MT120 25 21
MV120 23 15
FL181 52 51
FS186 48 28
SP187 4 9
FL191 31 29
No FNR 0 0
Activation was measured inJRG1728 ([Delta]lac [Delta]fnr) cells carrying FF(-71.5) or FF(-61.5) fused to lac in pRW50. Values are expressed as percentages of transcription activation by wild-type FNR. 100% activation = 2870 nmol/min/mg cell dry wt at FF(-71.5) and 2960 nmol/min/mg at FF(-61.5); 0%activation = 390 nmol/min/mg at FF(-71.5) and 280 nmol/min/mg at FF(-61.5). Data in the table are averages of three independent assays; in each case the standard deviation from the mean was <10%.

RESULTS

Isolation of positive control mutants of FNR

The first aim of this work was to identify single amino acid substitutions in FNR that result in a defect in transcription activation at Class I FNR-dependent promoters, but do not affect DNA binding or triggering by anaerobiosis. Following previous work with CRP (9 ,10 ), we reasoned that the location of such substitutions would identify the surface of FNR that interacted with [alpha]CTD during transcription activation. To identify such `positive control' mutants we adapted the strategy fully described by Bell and Busby (15 ). The starting point was the semi-synthetic FF(-71.5) promoter, which is completely dependent on FNR (5 ). This promoter, which contains a consensus DNA site for FNR centred between base pairs 71 and 72 upstream of the melR transcription start point (i.e. position -71.5), was cloned into plasmid pRW50 to give a FF(-71.5)::lac fusion. M182 ([Delta]lac fnr+) cells carrying the resulting recombinant score as Lac+ on indicator plates because chromosomally encoded FNR activates the FF(-71.5) promoter. To identify positive control FNR mutants we exploited plasmid pFNR, which carries the cloned fnr gene and is compatible with pRW50 derivatives. After mutagenesis of the cloned fnr gene by error-prone PCR, pFNR was transformed into M182 cells carrying the FF(-71.5)::lac fusion. Positive control mutants result in a Lac- phenotype, since they are unable to interact correctly with RNAP, and yet fold correctly and bind to DNA sites for FNR. Note that mutants that are unable to fold correctly will be unable to suppress activation of FF(-71.5) by the chromosomal fnr gene and will not be picked by this screen. After performing seven independent error-prone PCR mutagenesis reactions and screening >60 000 colonies, we selected 48 Lac- colonies apparently containing a trans-dominant pFNR derivative that interfered with activation of FF(-71.5) by wild-type FNR.


Figure 1. Three-dimensional model of an FNR monomer, without its extended N- and C-terminal domains, based on the known CRP structure taken from the Brookhaven protein database file 1CGP.PDB (a full discussion of the relation between the structures of CRP and FNR is to be found in ref. 2). The model shows the location of amino acid residues substituted in the FNR-positive control mutants described here. Residue D43 (red) is located on [alpha]-helix A, residues R72 and S73 (purple) are located on the loop between [beta]-sheets 3 and 4, residues T118 and M120 (blue) are located on [beta]-sheet 8, residue F181 (green) is located on the loop between [alpha]-helix D and [beta]-sheet 9 and residues F186, S187 and F191 (green) are located on [beta]-sheet 9.

Since FNR mutants defective in DNA binding or triggering by anaerobiosis would also have scored as Lac- in the first screen, we included a second screening step. This second screening, which was included to eliminate these types of mutants, exploited an FNR-repressible promoter, pndh. Thus, pFNR plasmid DNA isolated from each of the 48 Lac- colonies was transformed into strain JRG1728 ([Delta]lac [Delta]fnr) cells carrying a pndh::lac fusion in plasmid pRW50 and the fusion was used to assess the ability of each mutant to be triggered by anaerobiosis and to bind to DNA targets in vivo. We found that 20 of the 48 pFNR derivatives were defective in anaerobically induced repression of the pndh::lac fusion and were discarded. We concluded that the remaining 28 pFNR derivatives must encode positive control mutants of FNR: they are significantly defective in transcription activation at FF(-71.5), but are not defective in transcription repression at pndh (see below). The base sequence of each of these derivatives was determined and the amino acid sequence was deduced. This revealed that we had isolated 22 independent mutants (six of the mutants were obtained more than once from the same PCR reaction and were discounted). These 22 mutants were due to amino acid substitutions of nine different residues: D43, R72, S73, T118, M120, F181, F186, S187 and F191. The different substitutions are listed in Table 2 : in some cases substitutions were isolated in combination with changes at other positions, but these other changes were found to have little or no effect (see footnote to Table 2 ). Note that these substitutions include SF73, which we had previously found to interfere with transcription activation at a Class I FNR-dependent promoter (5 ). Figure 1 presents a model of the predicted structure of FNR, based on the crystal structure of CRP, showing that the nine substituted residues are all found on the same face of FNR. We conclude that this face must contain the amino acid sidechains that are important for transcription activation by FNR at the FF(-71.5) promoter. Figure 2 shows models of FNR bound to a DNA target site, again highlighting the locations of the different positive control substitutions that interfere with FNR-dependent activation of FF(-71.5).


Figure 2. Models of an FNR dimer bound to a target site in DNA viewed side-on and end-on. The models, based on the CRP-DNA structure presented by Schultz et al. (26), show the location of substitutions that interfere with activation at Class I FNR-dependent promoters, with colour coding as in Figure 1. Note that the patch identified by green colouring corresponds to activating region 1 of CRP (see Discussion). The patch identified by pink and blue colouring has no functional equivalent in CRP, but, whilst clearly distinct, is on the same face of FNR. Note that this patch is exactly in the direction of the path of the DNA, whilst the `green' patch (equivalent to activating region 1 of CRP) is off the path of the DNA.

Characterization of positive control mutants of FNR

The effects of the newly isolated substitutions in FNR on transcription activation at Class I promoters with the DNA site for FNR centred at positions -61.5 or -71.5 was determined. To do this, the different pFNR derivatives were introduced into strain JRG1728 ([Delta]lac [Delta]fnr) carrying the test promoters FF(-71.5) and FF(-61.5) fused to the lac operon in plasmid pRW50 and [beta]-galactosidase expression of cells grown anaerobically was measured. The data in Table 3 show that each of the substitutions causes similar substantial reductions in expression from both promoters, confirming the phenotypes that had been observed during the screen. We conclude that the same surface of FNR is likely to be involved during transcription activation at both promoters. To investigate the role of different amino acid sidechains in this surface, a number of different residues were replaced with alanine. R72, S73, G74, T118, M120 and S187 were selected, as these are located in three separate surface-exposed loops where the positive control substitutions had been isolated (Fig. 1 ). The results in Table 4 show that alanine substitution of S73 and G74 had no effect on transcription activation at FF(-71.5), whilst substitutions at R72, M120 and S187 have only marginal effects (<50%). In contrast, substitution of alanine at T118 greatly reduces activation, suggesting that the sidechain of T118 provides a crucial contact with RNAP. The lack of effect of the SA73 substitution suggests that the consequences of the SF73 substitution, previously reported by us, must be due to indirect effects: for example, the substitution of serine at position 73 by phenylalanine may generate a clash that hinders FNR-RNAP contacts. In control experiments we confirmed that the alanine substitutions had the same effect on activation at the FF(-61.5) promoter and that DNA binding was unaffected (as judged by repression of the ndh promoter; data not shown).

Table 4 Transcription activation by FNR carrying alanine substitutions
FNR derivative Activation at FF(-71.5) (%)
FNR 100
RA72 78
SA73 109
GA74 102
TA118 15
MA120 78
SA187 58
Activation was measured inJRG1728 ([Delta]lac [Delta]fnr) cells carrying FF(-71.5) fused to lac in pRW50. Values are expressed as a percentage of activation by wild-type FNR at FF(-71.5). The average activation values for wild-type FNR in nmol/min/mg cell dry wt was 2400. Each FNR derivative carrying an alanine substitution was shown to bind DNA normally, as measured by the ability to repress transcription activation from the FNR-repressible promoter pndh and all alanine scanning mutants were aerobically inactive, as measured in cells grown aerobically (data not shown).

Table 5 . Transcription activation by oriented heterodimers at FF(-71.5) derivatives
FNR derivatives   Promoter activities FY/YF
    FY(-71.5) YF(-71.5)  
Control experiment FNR/EV209 830 900 0.9
Heterodimer experiments SF73/EV209 2100 470 4.5
  MI120/EV209 2100 440 4.8
  SP187/EV209 1500 350 4.3
Activities were measured inJRG1728 ([Delta]lac [Delta]fnr) cells carrying FY(-71.5) or YF(-71.5) fused to lac in pRW50. Cells contained a pHW1 derivative encoding FNR carrying the EV209 substitution that permits FNR binding to the Y half-site. Cells also contained pFNR derivatives encoding either wild-type FNR or FNR carrying the SF73, MI120 or SP187 substitutions. Cells were grown anaerobically in L-broth supplemented with glucose, tetracycline, kanamycin and ampicillin. Promoter activities are expressed as [beta]-galactosidase activities (nmol/min/mg cell dry wt) and ratios were calculated from three independent sets of data.

Table 6 . Sequences of 27 RNA polymerase [alpha] mutants that interfere with the FF(-71.5) promoter
Amino acid substitution Codon substitution No. of isolates Activity of FF(-71.5)a (%)
EG261 GAA -> GGA 5b 70 ± 6
EK261 GAA -> AAA 1 61 ± 18
RC265 CGC -> TGC 2c 44 ± 11
NS268 AAC -> AGC 2 80 ± 11
LF300 CTT -> TTT 3 65 ± 13
LH300 CTT -> CAT 1 80 ± 17
EK302 GAG -> AAG 4d 61 ± 10
DG305 GAC -> GGC 1 60 ± 1
AD308 GCT -> GAT 2 57 ± 5
GA315 GGC -> GCC 1 77 ± 12
RP317 CGC -> CCC 1 66 ± 4
[alpha]-257+4 1 bp deletion in codon 258 1e 40 ± 2
[alpha]-259+7 2 bp deletion in codon 260 1e 69 ± 9
[alpha]-260 Stop at codon 261 1 33 ± 7
[alpha]-262 Stop at codon 263 1 22 ± 9
pLAW2 derivatives encoding [alpha] carrying the listed substitutions were isolated after mutagenesis of pLAW2 as described in the text. In each case the entire base sequence of the mutagenized segment of rpoA was determined.
aActivity was measured in vivo using M182 [Delta]lac fnr+ cells carrying a FF(-71.5)::lac fusion cloned in pRW50. These cells were transformed with pLAW2 derivatives encoding different [alpha] mutants and [beta]-galactosidase activities were determined. Activity values (± 1 SD) are expressed as percentages of activity with pLAW2 encoding wild-type [alpha]. Note that the observed reduction in expression must be an underestimate of the effect of each mutant since the experiment is performed with chromosomally encoded wild-type [alpha] subunits present.
bOne isolate contained a second amino acid substitution GS279 (GGT -> AGT). This second substitution has little or no effect on activation of FF(-71.5).
cOne isolate contained a second amino acid substitution QL283 (CAG -> CTG). This second substitution has little or no effect on activation of FF(-71.5).
dOne isolate contained a second amino acid substitution EG245 (GAG -> GGG). This second substitution has little or no effect on activation of FF(-71.5).
e[alpha]-257+4, a 1 bp deletion in codon 258, caused a frameshift and changed codon 262 to the stop codon TGA. The amino acid sequence is identical to wild-type [alpha] up to amino acid 257, with the addition of four different amino acids (X257GDLN). [alpha]-259+7, a 2 bp deletion in codon 260, caused a frameshift and changed codon 267 to the stop codon TAA. The amino acid sequence is identical to wild-type [alpha] up to amino acid 259, with the addition of seven different amino acids (Y259GIDCPLC).

Transcription activation by oriented heterodimers

Our results suggest that three separate surface-exposed loops in the FNR structure are involved in transcription activation at Class I FNR-dependent promoters. Since FNR is functional as a dimer, it is possible that each of these regions is functional in both subunits or in either the upstream or the downstream subunit. To investigate this we used the method of `oriented heterodimers', previously adapted for FNR by Bell and Busby (15 ). This method relies on the alteration of either the upstream or downstream half-site of the 22 bp FNR binding sequence at a target promoter from 5'-AAATTTGATGT-3' (designated F) to 5'-AAATTTAATGT-3' (designated Y). This creates the hybrid binding sites FY or YF, with the altered half-site located either downstream or upstream respectively, and these hybrid sites were incorporated into FF(-71.5) to give FY(-71.5) and YF(-71.5). Bell and Busby (15 ) showed that wild-type FNR is unable to recognize the altered half-site Y, whereas FNR carrying the substitution EV209 in the DNA binding helix is able to bind to Y. Thus, heterodimers between wild-type FNR and FNR EV209, which form in cells after introduction of two compatible plasmids encoding different fnr genes, bind to target promoters containing the hybrid binding sites with the FNR subunit carrying wild-type binding specificity binding to the F half-site and the FNR E209V subunit with altered DNA binding specificity occupying the Y half-site. In these experiments JRG1728 cells were transformed with plasmid pHW1, a pLG339 derivative encoding FNR EV209. These cells were further transformed with pFNR derivatives encoding FNR with wild-type binding specificity and the positive control substitution SF73, MI120 or SP187 (representative of substitutions in the three surface-exposed loops of FNR that we had identified). The data in Table 5 show that the three positive control substitutions in FNR all interfere with transcription activation at the hybrid promoter YF(-71.5) butdo not interfere with activation at FY(-71.5). Since the FNR subunit carrying the substitutions is targeted to the F half-site, we conclude that the activating region defined by the substitutions at S73, M120 and S187 are all functional in the downstream subunit at FF(-71.5).

Isolation of mutants in [alpha]CTD that interfere with a Class I FNR-dependent promoter

The second aim of this work was to identify single amino acid substitutions in the RNA polymerase [alpha] subunit that resulted in a defect in transcription activation at Class I FNR-dependent promoters. Following previous work with CRP (12 -14 ), we reasoned that the location of such substitutions would identify a surface in [alpha]CTD that interacted with FNR during transcription activation. To find such mutants we adapted the strategy described by Zou et al. (12 ), using M182 ([Delta]lac fnr+) cells carrying the semi-synthetic FF(-71.5) promoter cloned into plasmid pRW50 to give a FF(-71.5)::lac fusion. These cells score as Lac+ on indicator plates because chromosomally encoded FNR activates the FF(-71.5) promoter. To identify substitutions in [alpha]CTD that interfere with this activation we exploited plasmid pLAW2, which carries the cloned rpoA gene and is compatible with pRW50 derivatives. After mutagenesis of the segment of rpoA encoding [alpha]CTD by error-prone PCR, pLAW2 was transformed into M182 cells carrying the FF(-71.5)::lac fusion. After performing three independent error-prone PCR mutagenesis reactions and screening >70 000 colonies, we selected 27 Lac- colonies apparently containing a pLAW2 derivative encoding [alpha] that interfered with activation of FF(-71.5) by wild-type FNR. Each mutant pLAW2 derivative was isolated and the base sequence of the mutant rpoA gene was determined. Table 6 lists the changes found in each of the derivatives. Four of the mutant plasmids encode truncated [alpha]CTD, whilst the others carry single amino acid substitutions, the location of which identify sidechains likely to be involved in transcription activation at FF(-71.5). Figure 3 is a model of the structure of [alpha]CTD, showing the location of these sidechains.

DISCUSSION


Figure 3. A space-filling model of the predicted structure of [alpha]CTD, created from the Brookhaven protein database file 1COO.PDB, showing the location of the residues discussed here. Views (A)-(D) are related by a rotation of 90o around their y-axis. E261 is coloured red, R265 and N268 are coloured yellow and L300, E302, D305, A308, G315 and R317 are coloured blue (see Discussion for details).

Many bacterial transcription activators function by making a direct contact with the C-terminal domain of the RNAP [alpha] subunit. These activators bind upstream of the RNAP binding elements at target promoters and the role of the activator-[alpha]CTD interaction is to recruit RNAP to the promoter. In some cases the flexibility of the linker that anchors [alpha]CTD to the rest of the RNAP allows flexibility in the location of the activator binding site on the promoter DNA (reviewed in 8 ,20 ). Activators make contact with [alpha]CTD via surface-exposed patches (activating regions) that can be defined by the location of positive control substitutions. Similarly, the activation target on [alpha]CTD can be investigated by the location of substitutions that interfere with activation. However, to date there is no case where the details of activator-[alpha]CTD interactions are clearly understood.

Our results identify the activating region of FNR that interacts with [alpha]CTD. Interestingly, according to our model for the structure of FNR the activating surface contains three distinct adjacent surface-exposed loops (residues 71-75, 116-121 and 181-191; Fig. 1 ). These loops are located on a face of FNR that is distinct from the face that makes contact with the RNAP [sigma] subunit at Class II FNR-dependent promoters (see 5 ,15 ). Alanine scanning identifies T118 as providing the crucial sidechain for interaction with [alpha]CTD. Presumably the major contact is provided by the 116-121 loop and less crucial contacts are provided by the 71-75 and 181-191 loops (it is likely that D43 is not involved and that the effect of the DG43 substitution is indirect). Our results show that the same activating region is functional at promoters where the FNR site is located at positions -61.5 and -71.5 and is functional solely in the downstream subunit of the FNR dimer. In this respect, FNR and CRP are similar. Interestingly, however, the location and nature of the activating region that interacts with [alpha]CTD clearly differs between the two activators. In CRP the activating region that interacts with [alpha]CTD is confined to a single [beta] turn from residues 156 to 164 (9 ,10 ), corresponding to just one of the surface-exposed loops we have identified in FNR (the 181-191 loop; Fig. 2 ). However, our results clearly show that the major activating determinant in FNR is located in the adjacent 116-121 loop. Thus, whilst the structures of CRP and FNR may have been conserved, the precise nature of the activating region that interacts with [alpha]CTD differs. A possible explanation for this arises from the suggestion that the primary role of activator-[alpha]CTD interactions is `merely' to recruit RNAP to the promoter (21 ). We suppose that these interactions can be generated in many different ways and, thus, different activators `solve' their problems in different ways.

Our observation that deletions of [alpha]CTD interfere with expression from the FF(-71.5) promoter (Table 6 ) confirms that [alpha]CTD is essential for FNR-dependent transcription activation (note that these experiments were performed with plasmid-encoded [alpha] introduced in `trans' to wild-type [alpha]). We have also identified several single amino acid substitutions in [alpha]CTD that decrease expression at Class I FNR-dependent promoters (Fig. 3 ). The interpretation of these results is facilitated by the recent determination of a structure for [alpha]CTD (22 ,23 ) and by studies of the properties of several mutant [alpha] derivatives at UP element-dependent and activator-dependent promoters (see for example 12 -14 ). Expression from FF(-71.5) is reduced by substitutions at E261, R265, N268, L300, E302, D305, A308, G315 and R317. Tang et al. (13 ) concluded that E261 was the crucial residue for CRP-dependent transcription activation at the E.coli lac promoter, whilst Murakami et al. (14 ) claimed that the same surface of [alpha]CTD was involved in both DNA binding and in interaction with CRP at the lac promoter. Our screen identified substitutions both at E261 and in the DNA binding surface (R265 and N268) and, whilst it is possible that one or more of these residues does provide a direct contact with both CRP and FNR, it is also possible that the effects of these substitutions are indirect. Jafri et al. (24 ) reported that the EK261 substitution can interfere with activator-independent transcription initiation and we cannot, at present, exclude the possibility that DNA binding by [alpha]CTD is involved in the mechanism by which FNR activates transcription. The remaining substitutions (at L300, E302, D305, A308, G315 and R317) do not define a discrete `patch' but, rather, appear to cluster in two sub-regions (Fig. 3 ). It is likely that substitutions within this group identify the contact site for the activating region of FNR and, interestingly, in a previous study of the Salmonella typhimurium FNR equivalent, OxrA, Lombardo et al. (25 ) found that substitutions at positions 311 and 317 of [alpha]CTD interfered with OxrA-dependent activation of the S.typhimurium pepT promoter. Note, however, that it is unlikely that the L300, E302, D305, A308, G315 and R317 sidechains are all involved in contacts with FNR: some of the consequences of substituting these sidechains may be indirect and this will need to be resolved by alanine scanning and in vitro studies.

ACKNOWLEDGEMENTS

This work was funded by a BBSRC project grant and a BBSRC CASE studentship to S.M.W. We thank Jeff Cole, Richard Ebright, Jeff Green, John Guest and Patricia Kiley for many helpful suggestions and Christine Webster for expert technical assistance.

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*To whom correspondence should be addressed. Tel: +44 121 414 5439; Fax: +44 121 414 7366; Email s.j.w.busby@bham.ac.uk
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