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Nucleic Acids Research Pages 2075-2081  

Transcription activation by Escherichia coli FNR protein: similarities to, and differences from, the CRP paradigm
Introduction
Materials And Methods
Results
   Isolation of mutations that restore activity to FNR carrying a non-functional Activating Region 1
   Activity of FNR mutants
   Transcription activation by oriented heterodimers
   Effects of the EK47 and KR60 substitutions in FNR
Discussion
Acknowledgements
References

Transcription activation by Escherichia coli FNR protein: similarities to, and differences from, the CRP paradigm

Transcription activation by Escherichia coli FNR protein: similarities to, and differences from, the CRP paradigm

Bo Li, Helen Wing, David Lee, Hui-chung Wu, Stephen Busby*

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

Received January 28, 1998; Revised and Accepted March 10, 1998

ABSTRACT

During transcription activation at FNR-dependent promoters where the DNA site for FNR overlaps the -35 element, a surface-exposed activating region in the upstream subunit of the FNR dimer interacts with the C-terminal domain of the RNA polymerase [alpha] subunit. Starting with a cloned fnr gene encoding a defective FNR derivative carrying substitutions in this activating region, we screened a library of random mutations to identify substitutions that restored FNR activity. Activity can be restored by substitutions at residues T118, E47 and K60. The locations of these residues identify three separate surface-exposed regions of FNR that can play a role in transcription activation. These three regions appear to be analogues of Activating Region 1, Activating Region 2 and Activating Region 3 of the cyclic AMP receptor protein, CRP: our results underscore the similarities between FNR and CRP.

INTRODUCTION

The Escherichia coli CRP and FNR proteins are global activators of transcription initiation that regulate transcription from a large number of promoters in response to glucose and oxygen starvation respectively. The primary sequences of CRP and FNR are related: CRP and FNR are believed to have similar structures and to have evolved from a common origin (reviewed in 1,2). Both CRP and FNR activate transcription by making direct contact with RNA polymerase (RNAP) at target promoters. Binding sites for both CRP and FNR 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 (3,4).

The surface-exposed determinants of CRP that contact RNAP at different promoters have been studied in detail. At promoters where the DNA site for CRP is located upstream of the promoter elements for RNAP, a single 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 the C-terminal domain of the RNAP [alpha] subunit ([alpha]CTD: reviewed in 5,6). At promoters where the DNA site for CRP overlaps the -35 promoter element (and is centred near position -41, upstream of the transcript start), CRP functions by making two contacts with RNAP: Activating Region 1 of the upstream subunit of the CRP dimer interacts with [alpha]CTD, whilst a positively-charged surface-exposed patch in the downstream subunit of the CRP dimer (residues H19, H21 and K101, known as Activating Region 2) interacts with a contact site in the N-terminal domain of the RNAP [alpha] subunit ([alpha]NTD: reviewed in 7). At promoters where the DNA site for CRP overlaps the -35 element, CRP can also make a third non-native contact with RNAP that supplements the interactions made by AR1 and AR2. A surface exposed region in the downstream subunit of the CRP dimer (amino acids 52-58, known as Activating Region 3) interacts with a contact site near the C-terminal of the RNAP [sigma] subunit (reviewed in 7). This third non-native activating region was discovered from studies of amino acid substitutions that restore activity to CRP that had been inactivated by substitutions that destroy Activating Region 1 (8-10). These substitutions, which fall at K52 of CRP, unmask Activating Region 3 thereby creating a surface that can interact with RNAP. The locations of key residues in the three activating regions of CRP are shown in Figure 1A.


Figure 1. Models of CRP and FNR dimers bound to a target site in DNA viewed side-on. The models, based on the CRP:DNA structure presented by Schultz et al. (21), show the location of substitutions that affect transcription activation. The models were drawn using WebLab viewer version 2.0 (MSI). (A) Structure of a CRP dimer identifying H159 in Activating Region 1 (blue), H19, H21 and K101 in Activating Region 2 (red) and E58 in Activating Region 3 (yellow). Note that Activating Region 1 and Activating Regions 2 and 3 are displayed on the different adjacent faces of CRP subunits on one side of the CRP dimer. (B) Structure of an FNR dimer identifying T118 and S187 in Activating Region 1 (blue) and K60 and G85 in Activating Region 3 (yellow). Note that Activating Region 1 and Activating Region 3 are displayed on the different adjacent faces of FNR subunits on one side of the FNR dimer. (C) Structure of an FNR dimer identifying the locations where substitutions improve activation of FF(-41.5) by FNR carrying a defective Activating Region 1: T118 in Activating Region 1 (blue), E47 in Activating Region 2 (red) and K60 in Activating Region 3 (yellow).

The determinants of FNR that are involved in contacting RNAP at different promoters are less well understood. At promoters where the DNA site for FNR is located upstream of the promoter elements for RNAP, a surface of the downstream subunit of the FNR dimer interacts with a contact site in the C-terminal domain of [alpha]CTD (4,11,12). This surface of FNR, which is analogous to Activating Region 1 of CRP, includes residues T118 and S187. At promoters where the DNA site for FNR overlaps the -35 promoter element, FNR functions by making two contacts with RNAP: the determinant in the upstream subunit of the FNR dimer that is analogous to Activating Region 1 of CRP interacts with [alpha]CTD, whilst another surface-exposed patch in the downstream subunit of the FNR dimer, that appears to be the equivalent of Activating Region 3 of CRP, interacts with the RNAP [sigma] subunit (9,11; reviewed in 7). This Activating Region 3 homologue in FNR includes residues K60 and G85. The locations of some of the residues in the two activating regions of FNR are shown in Figure 1B.

Studies to date suggest that FNR does not contain a functional equivalent of Activating Region 2 of CRP. Thus, at promoters where the DNA site for the activator overlaps the -35 element, the downstream subunit of FNR contacts the RNAP [sigma] subunit (via Activating Region 3) whilst the downstream subunit of CRP contacts [alpha]NTD (viaActivating Region 2). However, since a single substitution in CRP can create an Activating Region 3 homologue able to interact with the RNAP [sigma] subunit (8-10), and because of the striking parallels between CRP and FNR, it was pertinent to investigate whether an Activating Region 2 homologue could be created in FNR. Thus, in this work, we have started with a mutant inactive FNR carrying a defective Activating Region 1 and searched for substitutions that restore activity. We report the properties of substitutions at three locations that appear to improve Activating Region 1 and Activating Region 3 of FNR and create an Activating Region 2.

MATERIALS AND METHODS

The E.coli [Delta]lac host strains used in this work were: M182 fnr+ and JRG1728 [Delta]fnr (4,12), and JCB387 and the narL narP derivative, JCB3884 (13). The plasmids and promoters used are listed in Table 1. By convention, promoter sequences are numbered with the transcript start as +1, with upstream and downstream sequences denoted by - and + prefixes, respectively. For measuring promoter activities, the different EcoRI-HindIII fragments carrying promoters were cloned into the low copy number lac expression vector, pRW50, to give promoter::lac operon fusions. To obtain single copy promoter::lac fusions, the method of Simons et al. (16) was used: strain JRG1728 was lysogenised with bacteriophage lambda derivatives carrying the different promoter::lac fusions.

Standard recombinant DNA, site-directed mutagenesis and sequencing technologies were used as in our previous work (4,11,12). The starting point was a pFNR derivative encoding FNR carrying the TA118 and SA187 substitutions that inactivate the Activating Region 1 homologue. Random mutations in this cloned fnr allele were created by exploiting Taq DNA polymerase, as in our previous work (11,12). Eight PCR reactions were performed, each containing 10 ng of pFNR template, 1 µM of each primer, 200 µM of each dNTP, 3 mM MgCl2 and Bioline Taq DNA polymerase. For screening, the resulting library of mutations was introduced by electroporation into JRG1728 cells carrying an FF(-41.5)::lac fusion cloned in pRW50. Transformants were plated on MacConkey lactose plates containing ampicillin and tetracycline, and Lac+ colonies were picked and purified. The pFNR derivatives were purified and the entire base sequence of the mutated fnr gene was determined in each case. In this study, we retained only those FNR derivatives that could activate transcription more efficiently at the FF(-41.5) promoter.

To determine the effects of different FNR mutants on transcription activation, pFNR plasmids encoding fnr derivatives were transformed either into JRG1728 cells carrying different promoters fused to the lac operon in pRW50, or into JRG1728 cells containing a single copy lysogen carrying the promoter::lac fusion. Cells were grown anaerobically in L-broth supplemented with 0.4% glucose and appropriate antibiotics. [beta]-Galactosidase activities were measured by the Miller method (17) as before: each reported activity value represents data from at least three independent measurements that differed by <10%. For oriented heterodimer experiments, two compatible plasmids carrying different fnr mutants were introduced into JRG1728 cells carrying the appropriate reporter promoter cloned in pRW50. FNR derivatives carrying the EV209 substitution in the DNA-binding recognition helix were encoded by pHW1, whilst FNR derivatives carrying a wild-type DNA-binding recognition helix were encoded by pFNR. [beta]-Galactosidase activities were measured in cells carrying both pHW1 and pFNR derivatives as above.

RESULTS

Isolation of mutations that restore activity to FNR carrying a non-functional Activating Region 1

JRG1728 [Delta]lac [Delta]fnr cells carrying an FF(-41.5)::lac fusion cloned in pRW50 score as Lac- on indicator plates as expression from the FF(-41.5) promoter is completely dependent on FNR. The introduction of plasmid pFNR, encoding wild type FNR, restores a Lac+ phenotype. Our previous work showed that Activating Region 1 of FNR is disrupted by the TA118 and SA187 substitutions (12). Thus, introduction of a pFNR derivative encoding FNR with the TA118 and SA187 substitutions results in a Lac- phenotype. To identify substitutions that improved the function of FNR we mutagenised this pFNR derivative and screened for mutations that restored the Lac+ phenotype.

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(-41.5) Semi-synthetic FNR-dependent promoter with FNR-binding site centred at position -41.5 upstream of the melR transcription start (4)
YF(-41.5) Derivative of FF(-41.5) with upstream half of FNR binding site, 5[prime]-AAATTTGATGT-3[prime] (designated F) changed to 5[prime]-AAATTTAATGT-3[prime] (designated Y) (4)
FY(-41.5) Derivative of FF(-41.5) with downstream F sequence replaced with Y sequence(4)
FF (-61.5) Semi-synthetic FNR-dependent promoter with FNR-binding site centred at position -61.5 upstream of the melR transcription start (4)
FF(-71.5) Semi-synthetic FNR-dependent promoter with FNR-binding site centred at position -71.5 upstream of the melR transcription start (4)
pnir7150 Escherichia coli nir promoter (13)
pnir7150 Escherichia coli nir promoter derivative with inactivated DNA site for NarL and NarP (13) H78.5
Plasmids
pRW50 Broad host range lac expression vector for cloning of different promoters on EcoRI-HindIII fragments: encodes resistance to 35 µg/ml tetracycline (14)
pAA121 General cloning vector for EcoRI-HindIII fragments derived from pBR322: encodes resistance to 80 µg/ml ampicillin (15)
pFNR Plasmid carrying fnr gene (and mutant derivatives) cloned in pBR322: encodes resistance to 80 µg/ml ampicillin (11)
pHW1 Plasmid carrying fnr gene (and mutant derivatives) cloned in pLG339: encodes resistance to 25 µg/ml kanamycin (4,11)

Table 2. Sequences of 13 fnr mutants that restore FNR activity
Candidate Codon position and
base substitution		 Amino acid
substitution		 No. independent
isolatesa
FNR TA118 SA187 KM60: 118: ACC to GCC Threonine to Alanine 3b
  187: TCC to GCC Serine to Alanine  
  60: AAG to ATG Lysine to Methionine  
FNR TA118 SA187 KR60 118: ACC to GCC Threonine to Alanine 4
  187: TCC to GCC Serine to Alanine  
  60: AAG to AGG Lysine to Arginine  
FNR TA118 SA187 EK47: 118: ACC to GCC Threonine to Alanine 4
  187: TCC to GCC Serine to Alanine  
  47: GAG to AAG Glutamic Acid to Lysine  
FNR TA118 SA187 EK47 KR60 118: ACC to GCC Threonine to Alanine 1
  187: TCC to GCC Serine to Alanine  
  47: GAG to AAG Glutamic Acid to Lysine  
  60: AAG to AGG Lysine to Arginine  
FNR TV118 SA187 118: ACC to GTC Threonine to Valine 1
  187: TCC to GCC Serine to Alanine  
pFNR derivatives encoding FNR carrying the listed substitutions were isolated after mutagenesis of pFNR TA118 SA187 as described in the text. In each case, the entire fnr base sequence was determined. The new changes resulting from this experiment are highlighted in bold text.
aIndependent isolates are defined as substitutions that arose in different PCR reactions.
bOne isolate also contained the DG97 substitution (GAC to GCC): this second substitution has no detectable effect on activation at FF(-41.5).

Activity of FNR mutants

The effects of the additional amino acid substitutions were measured at the FF(-41.5), FF(-61.5) and FF(-71.5) promoters. To do this, the different fnr derivatives were introduced into JRG1728 ([Delta]lac [Delta]fnr) cells carrying promoter::lac fusions and [beta]-galactosidase expression was measured (Table 3). In each case, promoter activity is completely dependent on FNR and is induced by growth in anaerobic conditions: [beta]-Galactosidase levels in cells grown aerobically are very low (data not shown). At the FF(-41.5) promoter, where the DNA site for FNR overlaps the -35 element, the TA118 and SA187 substitutions in Activating Region 1 reduce FNR-dependent expression to 26% of the control with wild type FNR: the residual activity is due to the Activating Region 3-RNAP contact. At the FF(-61.5) and FF(-71.5) promoters, where the DNA site for FNR is located further upstream, the substitutions in Activating Region 1 reduce FNR-dependent expression to background levels: the severe effect at these promoters is because only Activating Region 1 can make contact with RNAP. The results in Table 3 show that FNR-dependent expression at FF(-41.5) is increased by the substitution of valine at position 118, the substitution of lysine at position 47 and the substitution of methionine or arginine at position 60. Similar increases are observed when the FF(-41.5) promoter is located in single copy on the JRG1728 chromosome rather than on the plasmid pRW50. Interestingly, promoter activity is not increased further by combination of the substitutions at positions 47 and 60. The results in Table 3 show that the substitutions at positions 47 and 60 have no effect at the FF(-61.5) and FF(-71.5) promoters, where the DNA site for FNR is located further upstream. In contrast, substitution of valine at position 118 increases expression from both the FF(-61.5) and FF(-71.5) promoters.

Table 3. Transcription activation by `up' mutants of FNR
FNR derivative Activity at
FF(-41.5)		 Activity at
(%)chromosomal
FF(-41.5) (%)		 Activity at
FF(-61.5) (%)		 Activity at
FF(-71.5) (%)
    FF(-41.5) (%)    
Wild type FNR 100 100 100 100
No FNR 0 0 0 0
TA118 SA187 26 29 <1 2
TA118 SA187 KM60 71 82 <1 <1
TA118 SA187 KR60 66 73 <1 <1
TA118 SA187 KR60 EK47 64 75 <1 <1
TA118 SA187 EK47 62 78 <1 <1
TV118 SA187 62 69 29 16
JRG1728 ([Delta]lac[Delta]fnr) host cells, carrying different promoter::lac fusionsin pRW50 or a chromosomal FF(-41.5)::lac fusion as indicated, were transformed with different pFNR derivatives. Cells were grown anaerobically to mid-log phase in L-broth supplemented with 0.4% glucose and appropriate antibiotics and [beta]-galactosidase activities were measured in Miller units (nmol of hydrolysed ONPG/min/mg dry cell weight). Values in the table are expressed as percentages of transcription activation by wild type FNR. At FF(-41.5), 100% activation = 5663 U, 0% activation = 64 U; at the chromosomal FF(-41.5)::lacZ fusion, 100% activation = 3254 U, 0% activation = 38 U; at FF(-61.5), 100% activation = 1520 U, 0% activation = 138 U; at FF(-71.5), 100% activation = 2050 U, 0% activation = 375 U. Data shown are average at least three independent determinants that differ by no more than 10%.

Table 4. Transcription activation by oriented heterodimers
FNR derivatives [beta]-Galactosidase activity
(cloned in pFNR/pHW1) FY(-41.5) YF(-41.5) FY/YF
FNR/FNR EV209 1193 1346 0.89
FNR TA118 SA187/ FNR EV209 628 3703 0.17
FNR TA118 SA187/ FNR TA118 SA187 EV209 499 520 0.96
FNR TA118 SA187 KM60/ FNR TA118 SA187 EV209 651 1258 0.52
FNR TA118 SA187 EK47/ FNR TA118 SA187 EV209 577 1036 0.56
FNR TV118 SA187/ FNR TA118 SA187 EV209 651 476 1.37
[beta]-Galactosidase activities (Miller units) were measured in JRG1728([Delta]lac[Delta]fnr) cells carrying the FY(-41.5) or YF(-41.5) promoter fused to lac in pRW50. The cells also contained two compatible plasmids encoding FNR: pFNR derivatives encode FNR with a wild type DNA binding domain, and pHW1 derivatives encode FNR carrying the EV209 substitution in the DNA binding recognition helix. Cells were grown anaerobically to mid-log phase in L-broth supplemented with 0.4% glucose, and appropriate antibiotics. Ratios in the final column of the table were calculated from at least three independent sets of data. Note that valid comparisons can be made only between the activities of different promoters in the same genetic background (due to uncertainties in the relative expression and autoregulation of different FNR mutants).

Transcription activation by oriented heterodimers

We have investigated whether the substitutions at positions 47, 60 and 118 are functional in the upstream or downstream subunits of the FNR dimer, using the method of `Oriented Heterodimers', previously adapted for FNR by Bell and Busby (11). This method relies on the alteration of either the upstream or downstream half of the 22 bp FNR binding sequences at a target promoter from 5[prime]-AAATTTGATGT-3[prime] (designated F) to 5[prime]-AAATTTAATGT-3[prime] (designated Y). Wild type FNR is unable to recognise the altered Y half site, whereas FNR carrying the EV209 substitution in the DNA binding recognition helix binds preferentially to Y. In these experiments, two forms of FNR are co-expressed in the same cell and FNR heterodimers form. Thus, at the FY(-41.5) promoter with the hybrid FY FNR binding site, FNR heterodimers bind with the subunit with wild type DNA binding specificity on the upstream half site, and the subunit carrying the EV209 substitution on the downstream half site. In contrast, at the YF(-41.5) promoter with the hybrid YF FNR binding site, the subunit with wild type DNA binding specificity binds to the downstream half site, and the subunit carrying EV209 binds to upstream half site.

JRG1728 cells carrying a YF(-41.5)::lac or FY(-41.5)::lac fusion in pRW50 were transformed with a pHW1 derivative encoding FNR carrying the TA118 SA187 and EV209 substitutions. These cells were further transformed with pFNR derivatives encoding FNR with a wild type DNA binding recognition helix and various substitutions in the different activating regions. Data from this experiment, together with controls, are presented in Table 4. The results show that the EK47 and KM60 substitutions improve expression from the YF(-41.5) promoter compared to the FY(-41.5) promoter. Since FNR subunits carrying the EK47 and KM60 substitutions are targeted to the F half sites, we conclude that the increases in FNR-dependent activation caused by these substitutions are due to contacts involving the downstream subunit of the FNR dimer. In contrast, the TV118 substitution improves expression from the FY(-41.5) promoter compared to the YF(-41.5) promoter. This is consistent with the increase in FNR-dependent activation due to substitution of valine at position 118 being caused by contacts involving the upstream subunit of the FNR dimer.

Table 5. . Effects of the EK47 and KR60 substitutions in FNR FF(-41.5)H78.5
FNR derivative Activity at Activity at Activity at Activity at
  FF(-41.5) chromosomal pnir7150 pnir7150
Wild type FNR 5663 3254 1530 250
No FNR 64 38 51 16
EK47 6285 (x1.1) 4718 (x1.4) 2570 (x1.7) 1220 (x4.9)
KR60 7248 (x1.3) 4425 (x1.4) 2738 (x1.8) 1212 (x4.9)
pFNR derivatives were transformed into JRG1728 ([Delta]lac[Delta]fnr) host cells carrying different promoter::lac fusions, either cloned in pRW50 or as a chromosomal single copy. Growth conditions and assays were performed as described in the footnote to Table 3. [beta]-Galactosidase activities are expressed in Miller units and the factor increases due to the EK47 and KR60 substitutions are listed in brackets.

Effects of the EK47 and KR60 substitutions in FNR

The above results suggest that the EK47 and KR60 substitutions improve contacts between FNR and RNAP during transcription activation at FF(-41.5) and that this can compensate, at least partially, for defects in Activating Region 1. To investigate whether these effects were merely due to removal of the bulky E47 and K60 side chains we introduced alanines at these positions in FNR already carrying the TA118 and SA187 substitutions. Assays show that the EA47 and KA60 substitutions have very little effect on the activity of FNR carrying the TA118 and SA187 substitutions. We conclude that the improvements in transcription activation due to the EK47 and KR60 substitutions must be because of the presence of lysine at position 47 or arginine at position 60. Thus, we investigated the effects of the EK47 and KR60 substitutions alone on FNR activity in the presence of a fully functional Activating Region 1 (i.e. in the absence of the TA118 and SA187 substitutions). The results in Table 5 show that the EK47 and KR60 substitutions cause only marginal increases in expression of the FF(-41.5)::lac fusion. Because these effects were unexpectedly small, we repeated the experiment using a second promoter, the promoter of the E.coli nir operon, that is also dependent on FNR binding to a DNA site overlapping the -35 element. However, expression from this naturally-occurring promoter is co-dependent on both FNR and the activators NarL and NarP (13). Table 5 shows the effects of the EK47 and KR60 substitutions on expression from the wild type nir promoter, cloned on the pnir7150 fragment, and from a nir promoter derivative, H78.5, in which the DNA site for NarL and NarP is misplaced and inactivated. The results show that, whilst the EK47 and KR60 substitutions cause a 1.6-1.8-fold increase in expression from the wild type nir promoter, they increase expression by 4-5-fold with the H78.5 derivative. Complementary experiments with the wild type nir promoter in a narL narP background (strain JCB3884) show a similar effect (data not shown). From these results we conclude that the EK47 and KR60 substitutions in FNR have more significant effects when some other aspect of transcription activation is defective. Thus, at the FF(-41.5) promoter, clear effects are seen only when Activating Region 1 of FNR is defective. At the nir promoter, large effects are seen when there is a defect in the function of the co-activators, NarL and NarP, due to inactivation of the DNA site for NarL and NarP (as at the H78.5 derivative), or due to mutations in the narL and narP genes.

DISCUSSION

This work has shown that substitutions at positions 118, 47 and 60 in FNR can compensate for defects in Activating Region 1 of FNR. Position 118 falls within Activating Region 1 of FNR and thus the substitution of valine at this position must be improving the function of Activating Region 1, that makes contact with [alpha]CTD during transcription activation. Consistent with this, the effect of the valine substitution at position 118 is mediated by the upstream subunit of the FNR dimer at the FF(-41.5) promoter. Furthermore, this substitution also increases transcription activation at the FF(-61.5) and FF(-71.5) promoters where the DNA site for FNR is located further upstream.

Figure 1 shows that the location of position 47 in FNR falls in a region that is equivalent to Activating Region 2 of CRP: since FNR has a 29 amino acid N-terminal extension, the equivalent residue in CRP is at position 18. We suggest that the effects of the EK47 substitution in FNR are due to the creation of an Activating Region 2 homologue in FNR. Consistent with this, our study shows that the presence of a positively charged residue, Lysine, at position 47 is essential for the function of this activating region. Recall that the function of Activating Region 2 of CRP is also dependent on positively charged side chains: substitutions that replace H19, H21 and K101 of CRP with negatively charged amino acids inactivate Activating Region 2, whilst substitutions that replace E96 with a positively charged amino acid improve Activating Region 2 (7,18). Also consistent is the observation that the effects of the EK47 substitution are caused by the downstream subunit of the FNR dimer at the FF(-41.5) promoter, and that the EK47 substitution has no effect at the FF(-61.5) and FF(-71.5) promoters.

Figure 1 shows that position 60 in FNR falls in a region that is equivalent to Activating Region 3 of CRP. The effects of the KR60 and KM60 substitutions in FNR appear to be due to the improvement of the Activating Region 3 homologue in FNR. Consistent with this, we previously showed that other substitutions at positions 60 and 61 of FNR reduced transcription activation at the FF(-41.5) promoter (11). Also consistent is the observation that the effects of the KR60 and KM60 substitutions are caused by the downstream subunit of the FNR dimer at the FF(-41.5) promoter, and that these substitutions have no effect at the FF(-61.5) and FF(-71.5) promoters.

The substitutions in FNR described in this work identify three potential activating regions of FNR. At CRP-dependent promoters where the DNA site for CRP overlaps the -35 region, transcription activation is due to interactions involving Activating Region 1 and Activating Region 2: a third region, Activating Region 3, can be unmasked by a simple substitution. Likewise, for FNR, at promoters where the DNA site for FNR overlaps the -35 region, transcription activation is due to interactions involving Activating Region 1 and Activating Region 3: a third region, Activating Region 2, can be unmasked by a simple substitution. Our results underline the similarity between FNR and CRP. It is now apparent that FNR and CRP belong to a large family of transcription factors that are presumed to share common structures and to activate transcription by similar mechanisms (1,2). We suppose that different members of the family use different combinations of the three available activating regions to interact with RNAP, according to the sequence and organisation of the target promoters at which they act. For example, HlyX, the FNR homologue of Actinobacillus pleuropneumoniae, carries an enhanced Activating Region 1 (19,20).

Our work shows that the function of inactive FNR carrying a defective Activating Region 1 at the FF(-41.5) promoter can be restored by substitutions that improve the function of any of the three activating regions. Interestingly these substitutions have but small effects in wild type FNR, presumably because wild type FNR is already an efficient activator of transcription initiation at this promoter. Thus, improvements of Activating Region 2 and Activating Region 3 have substantial effects at the FF(-41.5) promoter only when Activating Region 1 is disabled. Similarly, large effects are only seen at the nir promoter when there is a defect in the function of the co-activators, NarL and NarP. Presumably the effects at any promoter depend on the functional role of each contact and the way that the promoter is organised: these details remain to be determined.

ACKNOWLEDGEMENTS

This work was funded by a BBSRC project grant and a Chinese Government scholarship to Bo Li. We are greatly indebted to Andrew Bell, Jeff Cole, Richard Ebright, Jeff Green, John Guest, Charles Miller and Nigel Savery for many helpful suggestions throughout this work.

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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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