Phosphorylation of the Rhizobium meliloti FixJ protein induces its ...

THE

JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 269, No. 38, Issue of September 23, pp. 23784-23789, 1994 Printed in U.S.A.

Q 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Phosphorylation of the Rhizobium meZiZoti FixJ Protein Induces Its Binding to a Compound Regulatory Region at the fixK Promoter* (Received for publication, May 24, 1994, and in revised form, July 1, 1994)

Anne GalinierS, Anne-Marie Garnerone, Jean-Marc ReyratQ,Daniel Kahn, Jacques Batutn, and Pierre Boistard From the Laboratoire de Biologie Moliculaire desRelations Plantes-Microorganismes, INRA-CNRS, B.P 27, 31326 Castanet-Tolosan Cedex, France

The FixJ proteinis a member ofthe regulatorclass of molecular weight phosphate donor such as acetyl phosphate,in two-component systems involved in the transcriptional the absence of FixL, resulted in a strong increaseof FixJ tranactivation of nitrogen fixation genes inRhizobium m e - scriptional activity as measured in an i n vitro transcription ZiZoti. Phosphorylation of FixJ was previously demon- assay. However, incubation of FixJC in the presence of acetyl strated to dramatically enhance its transcriptional ac- phosphate had no effect on its transcriptional activation (8). nifA and fixK promoters. Here we show that tivity at the This constitutive activity of FixJC suggests that the aminothe isolated carboxyl-terminal domain of FixJ, FixJC, terminal regulatory domain represses the activity of the output binds the fixK promoter, whereas binding of the fulldomain in the unphosphorylated FixJ protein and that this length FixJ protein requires its phosphorylation. By inhibition can be relieved upon phosphorylation (7, 8). analyzing the DNase I and Exonuclease I11 protection In this study, we demonstrate that the isolated carboxylpatterns of the wild-type and a mutantfixK promoter, terminal domain of FixJ binds DNA whereas binding of the we have identified two overlapping binding regions for both phosphorylated FixJ and FixJC. A higher affinity full-length protein depends on its phosphorylation. Exonucle-69 and -44 relative ase I11 and DNase I protection experiments at t h e fixK proregion is located between positions to the transcription start site, and a lower affinity re- moter ( p f i x K )by FixJC and phosphorylated FixJ have led to gion, between positions -57 and -31, overlaps the -35 the identification of two overlapping protected regions: onelocated between positions -69 a n d -44 from the transcription region of the promoter. start site and another located between positions-57 a n d -31. We provide evidence that phosphorylated FixJ binds these two The soil bacterium Rhizobium meliloti fixes atmospheric ni- regions simultaneously. A model for the respective rolesof t h e trogen inside alfalfa root nodules. Nitrogen fixationgenes (nif two regions will be presented. a n d f i x ) undergo a cascade regulation since their expression EXPERIMENTALPROCEDURES depends on two regulatory genes, nifA and fixK, whose expresProtein Purification-FixJ and FixJC (a kind gift of S. Da Re and J . sion is itself under the controlof the pair of regulatory genes ) phosFourment) were purified as described (8, 10). FixJ (100 p ~ was totwo-component fixL and fixJ (1,2). FixL and FixJ belongthe phorylated in the presence of 20 mM acetyl phosphate (Sigma), as defamily of regulatory proteins which, in most of the cases, pro- scribed previously (10, ll). mote transcription of target genes in response to specific enviIn Vitro Dunscription Assays-The plasmid templates used for asronmental signals (2, 3). FixL carries a heme-binding oxygen- saying in vitro transcription from the wild-type fizK promoter and its sensing domain and a carboxyl-terminal domain homologous to mutant allele (pJMR300 and pJMR301, respectively)are derivatives of the carboxyl-terminal kinase domain of the receptor compo- pTE103 (12) which contains a multiple cloning site positioned between nents (2, 4-6). FixJ carries an amino-terminal phosphorylat- two strong transcriptional terminators. pJMR300 results from the cloning of a 795-bp' EcoRI-BamHI fragment containing the fWcK promoter in able receiver domain characteristic of all regulator components pTE103 (10).pJMR301 wasconstructed as follows: a 617-bp EcoRI-Hina n d a carboxyl-terminal domain, also called output domain, dIII fragment was excised from the M13 derivative, M13FW63 (-63 responsible for transcriptional activation (2, 7) (Fig. lA). In- GAAT)(13).The ends of this fragment were made blunt using the Klenow deed, a protein consistingof the sole carboxyl-terminal domain, fragment of DNApolymerase I andligated into HincII-digested pTElO3. Single round transcription assays were performed as described (10) FixJC, is sufficient for the activation of the targetnifA and fixK in thepresence of various concentrations of FixJC or FixJ proteins and promoters in vivo (7) a n d i n vitro (8). Under low oxygen conpJMR300 or pJMR301 DNA as a template. In this assay, transcription centrations, as in the nodule environment, FixL promotes FixJinitiates at the genuine fizK promoter and stops at thedownstream T7 phosphorylation, thus allowing nifA and fixK transcription, terminator thus leading to a transcript of a specific size. By construcsince phosphorylation of FixJ dramatically enhances its tran- tion, the size of the fizK transcript generated from pJMR301 is larger scriptional activity (9, 10). Phosphorylation of FixJ from a low than that generated from pJMR3OO (390 nt uersus 370 nt). Gel Retardation and DNase I Footprinting Experiments-A HincII* This work wassupported in part by an EEC grant in the framework BamHI 319-bp fragment containing the wild-type fizK promoter (Fig. of the BIOTECH Program and AIP INRA Microbiologie. The costs of 1B) was prepared from plasmid pJMR300 and 3’ end-labeled a t the Amersham Corp.) using the publication of this article were defrayed in part by the payment of page BamHI site by [a-32PldATP (3000 Ci/mmol, charges. This article must therefore be hereby marked “aduertisement” Klenow fragment of DNA polymerase I. The labeled fragment was purified from free nucleotides by Sephadex G-50 chromatography. in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Gel retardation assays were performed in a total volume of 10 pl. A $ Supported by an EEC grant. 0 Supported by a doctoral fellowship from the Ministere de la Re- typical assay mixture contained 43 mM Tris acetate (pH 8.0), 30 mM cherche et de l’Enseignement. Present address: Unit4deGBnetique potassium acetate, 8 mM magnesium acetate, 27 mM ammonium aceMycobactkrienne, Institut Pasteur, 28, Rue du Docteur Roux, 75015 tate, 1 mM dithiothreitol, 80 mM KCl, 10% glycerol, 4% polyethylene Paris, France. glycol, 100 pg/ml bovine serum albumin, 25pg/mlpoly(dI.dC)ll To whom correspondence and reprint requests should be addressed. Tel.:33-61-28-50-54; Fax: 33-61-28-50-61; E-mail: batut@toulouse. The abbreviations used are: bp, base pair(s); nt, nucleotide(s). inra.fr.

23784

FixJ DNA Binding

23785

A poly(d1,dC) as bulk carrier DNA, radioactive DNA probe (25,000 cpm) and FixJC protein. After 10 min of incubation a t 30 "C, the samples were loaded onto a native6% polyacrylamide gelat high ionic strength 4 * (14) and electrophoresed for 1 h a t 10 V/cm. Radioactive compounds 1 FixJN 130 FixJC 204 amino-acids were detected by autoradiography after overnight exposure to output regulatory X-Omat-AR Fuji film at room temperature. domain domain The DNase I footprinting method of Galas and Schmitz (15) was used. FixJ or FixJC was incubated with the 3' end-labeled DNA probe B Fragment used for DNase I footprinting for 10 min at 30 "C in 45 plof the binding buffer describedabove. The mixture was treated for 1 min at the same temperature with 2 ng of Hincll BamHI bovine pancreas deoxyribonuclease I (Pharmacia), after adjusting CaCl, 5' and MgCI, concentrations to 5.5 mM and 55 m, respectively. DNase I 5' 3' digestion was stoppedby addition of EDTA (20 mM final concentration). -249 319-b~ +1 +62 DNA was precipitated with ethanol in the presenceof 400 mM sodium acetate and 20 pg of glycogen, washed with 70% ethanol, and dried. Fragments used for Exo 111 protection experiments DNA was dissolved in sequencing samplebuffer. After 5 min at 70 "C, the samples (4 pl, -20,000 cpm) were electrophoresed in a denaturing BamHI Hind I11 6% polyacrylamide gel (16).Dry gels were autoradiographed overnight 5 5' a t -80 "C with an intensifying screen. Exonuclease 111Protection Experiments-For Exonuclease I11 experiments, the wild type fizK promoter and its mutant derivative were EcoRI BssHII 5 recloned in pBluescript (Stratagene). pAMGlOO plasmid results from 5' the cloning of a 319-bp BamHI-HincII fragmentfrom pJMR300 (10)in -102 + 1 1-62 pBKS' previously digested with EcoRV and BamHI. pAMG2OO was 286-bp constructed by excising the 348-bp HincII-PstI fragment from pJMR301 and ligating it into pBSK+previously digested withthe EcoRV and PstI FIG.1. Structure of FixJ (A) and of the Fzg promoter regions analyzed in this study ( B ) .The structure of FixJ is as indicated in restriction enzymes. Kahn and Ditta (7). The fwK promoter sequences are represented as Both pAMGlOO and pAMG2OO plasmidswerefirstdigested by BamHI for the top strand analysis orby HindIII for the bottom strand hatched boxes to distinguish them from vector DNA. The sequence analysis. After dephosphorylation of the free ends with bacterial alka- CTAA from positions -66 to -63 in the wild-type promoter has been at the Hind111 site for the replaced by GAAT in the mutantFW63 promoter (13). line phosphatase, the plasmids were cleaved top strand analysis or at the BamHI site for the bottom strand analysis (see Fig.1 B ) . The 323-bp HindIII-BamHI fragments were purified from Unphosphorylated FixJ did not protect the fixK promoter a n agarose gel using the Jet Sorb kit (Bioprob system) and 5' endlabeled at the BamHI sitefor the top strand analysis or at the HindIII from DNase I digestion even a t concentrations upto 48 p~ (Fig. site for the bottom strandanalysis by [ys2P1ATP (3000 CUmmol, 2). However, FixJ createda hypersensitive siteon fixK DNA at Amersham Corp.) using T4polynucleotide kinase. position -62 relative to the transcription start site. "he intenThe DNA probe for the bottom strand analysis of the mutant prosity of the resulting band increased with FixJ concentration. moter was prepared as follows. A DNA region including the mutant This first suggested an interactionbetween FixJ and DNA. promoter was specifically amplified from pAMG2OO by the polymerase The phosphorylated FixJ preparation induced the presence chain reaction technique using both M13 reverse primer and anoligoof a hypersensitive siteat position -62, as already observed for nucleotide ("TATGAAWCCTCGGCGTGATCAACA3') containing an unphosphorylated FixJ, althougha t a lower protein concentraEcoRI site at its 5' end. The polymerase chain reaction product was digested by BssHII, dephosphorylated with bacterial alkaline phospha- tion (Fig. 2). Moreover, protection of the fixK promoter region tase and then cleaved at the EcoRI site(seeFig. 1B).The 286-bp around position -62 was observed at a 12 PM concentration of BssHII-EcoRI resulting fragment was purifiedfrom an agarose gel as the phosphorylated FixJ preparation whereas no protection described above, and 5' end-labeled at the BssHII site by [Y-~~PIATP wasdetected at a 48 concentration of unphosphorylated (3000 CUmmol, Amersham Corp.) using T4polynucleotide kinase. The ExonucleaseI11 protection experiments were carried out accord- FixJ (Fig. 2). Further experiments showed that protection by ing to the method of Shalloway et al. (17). A typical assay mixture FixJ-PO, actually extends from position -69 down to position contained 20 mM Tris-HC1 (pH 8.01, 10 m~ 0-mercaptoethanol, 6 mM -30 (Fig. 3B, lane 3) although protection was slightly weaker in magnesium chloride, 0.1m EDTA, 100 mM KCl, 10% glycerol, 20 pg/ml as reproducibly detected (compare the -30 region and, thus, not poly(dI.dC)-poly(dIdC),radioactive DNA probe (25,000cpm), and Fig. 2 and Fig. 3B). DNase I footprinting experiments on the purified protein. After a 5-min incubation at 30 "C, the mixture was protected region as for the bottom treated for 2.5 min at the same temperature,a in finalvolume of 70 pl, top strand revealed the same with 260 units of Escherichia coli Exonuclease I11 (Life Technologies, strand, from position -69 to -30; however, no hypersensitive Inc.). Digestion was stoppedby addition of EDTA (20 mM final concen- site to DNaseI was detected (data notshown). tration)and phenoVchloroform extraction.Thesampleswerethen We conclude from the above data that FixJ isa DNA-binding treated and analyzed on a denaturing gel as described above. protein whose ability t o bind DNA is dramatically enhancedby phosphorylation. The IsolatedCarboxyl-terminal Domain of FixJ Binds Phosphorylation of FixJ Enhances Its Binding to DNA-FixJ DNA-Previous studies have shown that the isolated carboxylcontains a putative helix-turn-helixmotif in itscarboxyl-termi- terminal domainof FixJ, FixJC,is active in transcription, both proposed to be a DNA- in vivo and in vitro, without being phosphorylated (7, 8). We nal domain(Fig. lA)and has thus been binding protein (2,7). However, no experimental evidence sup- thus predicted that FixJC should bind the fixK promoter as of FixJ efficiently as FixJ-PO, does. This was first tested ina gel shift ported this prediction so far.Sincephosphorylation strongly stimulatesits ability to activate transcription,we hy- assay. Whereas neither FixJ nor FixJ-PO, did stably complex to pothesized that one effect of phosphorylation could be to pro- pfixK (although some smearing was observed with FixJ-PO,, mote FixJ binding toDNA. We thus tested the abilityof puri- data notshown), a complex was observed with FixJC (Fig. 3.4). fied FixJ either unphosphorylated or phosphorylated (FixJ- The binding site of FixJC was mappedby DNase I footprinting. PO,) to protect from DNase I attack a DNA fragment that was As anticipated, FixJC, which contains the putative helix-turnknown from previous work (13) to contain a functional fixK helix motif, bound DNA in the sameregion as FixJ-PO, since it promoter (Fig. 1B). FixJ was phosphorylated by incubation protected pfixK from DNase I attack in the-69 t o -44 region with acetyl phosphate under conditions that we knew to result (Fig. 3B). However, thefootprintgenerated by FixJCwas in phosphorylation of about 20% of FixJ monomers (10). smaller than thatobserved with FixJ-PO, since no protection RESULTS

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23786

FixJ-PO4

FixJ

FixJ DNA Binding A-

0 40 8 0.8 0.08 pM FixJC

I 6-

B-

-62

I

-30

I

FixJ-PO4 binding region

FIG.2. Effect of FixJ phosphorylation on DNA binding. The 319-bp end-labeled fragment (bottom strand analysis) was incubated with decreasing amounts of FixJ-PO, or FixJ and subsequently submitted to DNase I digestion. The DNase I hypersensitive site is indicated FIG.3. Binding of FixJC to the fi-t.K promoter. A, gel retardation by a n arrow. of a pfixK fragment by FixJC. The 319-bp end-labeled fragment containing pfmK was incubated with various amounts of pure FixJC. B, was observed between positions -44 and -30, even a t very high mapping of the FixJC-binding region on the fucK promoter by DNase I footprinting. The 319-bpend-labeledfragmentwasincubatedwith FixJC concentrations (up to200 PM, data not shown). FixJC (lane2,60 PM), FixJ-PO, (lane 3,40PM), and FixJ(lane 4,80 PM); Among possible reasons for FixJ-PO, and FixJCyielding dif- lane 1, DNA alone. The DNase I hypersensitive site is indicatedby a n ferent DNase I footprints, we considered the simple one that arrow.

FixJ-PO, being larger than FixJC mayprotect the -35 region from DNase Iattack by steric hindrance rather than by making ever, no difference could be detected in the efficiency of transpecific contacts to DNA. We reasoned that, if the above model scription from the wild type and mutant promoter (Fig. 4B). were true, binding to the upstream (-69, -44) region should be This clearly indicates that binding of FixJC to the (-69, -44) essential for activation of transcription by both FixJC and FixJ- region is not essential for activation of transcription in vitro. PO,. Consequently, mutations in the (-69, -44) region that FixJC Binds pfiK at ltvo Different Regions-In order to prevent FixJ-PO, or FixJC bindingshould render thefixK pro- account for the ability of FixJC and FixJ-PO, to activate the moter inactive in transcription. mutant fixK promoter, we hypothesized that these proteins The FixJ-binding Region Located between Positions -69 a n d were able tobind the -35 region of the mutantpromoter, even -44 Is Not Essential for fixK Dunscription inVitro-A mutant though this was notevidenced by the DNase I technique. We first reappraised theexistence of a binding site for FixJC fixK promoter in which 3 bases at position -66, -65, and -63 have been changed (pFW63in Waelkens et al. (13))was tested in the -35 region of the wild-type fixK promoter using the for binding of phosphorylated FixJ or FixJC ina DNase I foot- Exonuclease I11 (Exo 111) protection technique essentially beprinting assay. No protection of the mutant fixK promoter by cause of its higher sensitivity as compared to the DNase I either FixJC (up to 200 PM)or phosphorylated FixJ (up to 80 technique and also because some DNA-protein complexes are known to be disrupted by DNase I. p ~ was ) observed and the hypersensitive bandat position -62 FixJ did not protect the fixK promoter fragment from Ex0111 was notdetected (data notshown). Thus, the mutant promoter is defective in FixJ-PO, and FixJC binding. digestion (data not shown), thus reinforcing the evidence that Transcription from the wild-type and mutantpromoters was FixJ bindsDNAvery poorly. In contrast,Exo I11 was blocked a t compared in response to increasing amounts of FixJ-PO, and position -31 on the top strand(Fig. 5A) and atposition -70 on FixJC in an in vitro assay (10). In this assay, characteristic the bottom strand (Fig. 5B) following incubation with phosphotranscripts of 370 and 390 nt are synthesized from the wild rylated FixJ. Thus,Exo I11 and DNase I footprinting analyses type and mutant promoters, respectively (see “Experimental are fully consistent for FixJ-PO, since in both cases FixJ-PO, entirely protects a region located between -69 and -31. Procedures”). By contrast, FixJC protected the top strand from Exo I11 Maximal activation of the mutant fixK promoter was obtained at 30 p~ phosphorylated FixJ insteadof 5 PM for the wild digestion at two specific sites corresponding to bases -31 and type promoter (Fig. 4A). Thus, mutations that prevent, or a t -44 of the fixK promoter (Fig. 5A). The presence of two distinct least stronglyreduce, binding of FixJ-PO, to thepromoter had bands resistant toExo I11 digestion most likely arose from the a down effect on transcription thatcould be compensated for by fact that a significant part of the DNA molecules were solely a 6-fold increase in FixJ-PO, concentration. With FixJC, how- occupied at the upstream site at moderate concentrations of

F i d DNA Binding A-

FIG.4.In vitro transcription assay from the wild-type and the FW63 mutant fixK promoter in response to increasing concentrations of FixJ-PO, (A) or FixJC ( B ) .The transcripts initiated a t the fixK promoter are indicated by arrows. The size of transcripts generated from the wild-type and mutantpromoters are 370 nt and 390 nt, respectively.

FixJ-PO4

FixJC

H

*

B- wild type mutant

mutant

PfiXK

pfxK pfixK

l

0 2.5 5 7.5 15 30 0 2.5 5 7.5 15 30 pM FixJ-PO4

1

B- Bottom strand

A- Top strand 4

b

wild type PfXK

23787

-

FixJ-PO4 FixJC

c1 I02040 0 I O 20 40 pM FixJC

-7

c m m m y-mmm

A- Top strand

-

B- Bottom strand

FixJ-PO4 FixJC

FixJ-PO4 FixJC

-Ex0 0 10 60 :IO utM l ” L

-E~OO2010526030105 PM

l i l ”.

*-a

- -57

.-3 1

I

FIG.5. Mapping of the FixJC and FixJ-PO, binding regions on the wild-type fixK promoter by Exonuclease I11 protection experiments. A, top strand analysis. B, bottom strand analysis. The 323-bp end-labeled fragments were incubated with decreasing amounts of FixJ-PO, or FixJC as indicated. The bands marked by asterisks in panel B correspond to Exo I11 blocks a t -31 and -44 positions on the top strand. Their detection in this experiment is accidental and wasshown to result from a contaminating labeling of the top strand.

FixJC (30 PM). Increasing FixJC concentration to 60 PM resulted in the -31 band becoming the major band (Fig. 5 A ) . Analysis of the bottom strand revealed only one band, corresponding to position -70, on the promoter even a t 2 p~ FixJC (Fig. 5 B ) . The inability to detect the -35 binding site in this case probably reflects the higher affinity of the (-69, -44) region for FixJC so that molecules that are occupied at the -35 site are also occupied at the (-69, -44) position. Thus, these data are consistent with two binding regions for FixJC: a higher affinity region located between positions -69 and -44 and a region of lower affinity extending until position -31. We then carried outExo I11 protection experiments with the mutant fixK promoter and either FixJC or phosphorylated FixJ (Fig. 6). FixJ-PO, and FixJC yielded similar protection patterns when analyzing the top strand (Fig. 6 A ) since a single band at position -31 was now observed with both proteins. Hence, the mutations abolished binding of FixJC to the (-69, -44), region thus confirming the evidence obtained from DNase I footprinting. However, binding in the -35 region was observed with both FixJC and FixJ-PO,. This result is thus consistent with our prediction that occupancy of the -35 site is required

FIG.6. Mapping of the FixJC and FixJ-PO, binding regionson the mutant FW63 fixK promoter by Exonuclease I11 protection experiments. The 323-bp fragment and the 286-bp fragment (see Fig. 1B)were used for the top strand (A) and for the bottom strand analysis ( B), respectively.

for transcription of the mutantfixK promoter by FixJC or FixJPO,. Analysis of the bottom strand (Fig. 623) showed the absence of the -70 border and instead indicated a protection corresponding to position -57 for both with FixJC and FixJPO,. Altogether these data are consistent with existence the of two binding regions for FixJ-PO, located between positions (-69, -44) and (-57, -311, respectively. DISCUSSION

In vitro experiments have shown that FixJ is a transcriptional activatorwhose activity is enhanced by phosphorylation (9, 10). In this paper we present data showing that phosphorylation enhances FixJ binding to the fixK promoter, thereby accounting, in a simple model, for the enhancement of FixJ transcriptional activity. The isolated carboxyl-terminal domain of FixJ, FixJC, activates nifA and fixK promoters constitutivelyin vivoand in vitro (7,8).In orderto account for the constitutivity of FixJC, Kahn and Ditta (7) proposed that the amino-terminal receiver domain of FixJ has an inhibitory effect on the activity of the output domain that is relieved by phosphorylation. We show here that FixJC, contrary to FixJ, binds the fixK promoter to the same extent as phosphorylated FixJ. Since the carboxylterminal domain of FixJ carries a helix-turn-helix motif (2)

23788

FixJ DNA Binding 100%

i ’

0%

i ‘ -3s

1

FIG.7. Localization of the two FixJ-PO, protected regionson the R. meliloti mK promoter. The regions protected by FixJ-PO, are indicated as gray boxes. The upstream binding region is represented by a larger box to indicate, on a qualitative ground, its higher affinity as compared to the downstream binding region. The DNase I hypersensitive site is indicated by an arrowhead. The activity of two 5’-deleted f i K promoters, as measured in vivo in R. rneliloti, is normalized to the full-length promoter (13,18).Arrows indicate the 5‘ ends of these deleted promoters.

likely responsible for DNA binding, our data are in agreementthe two binding regions. Consistent with this model and with with a model in which the nonphosphorylated receiver domain the independent binding of FixJC to the two binding regions prevents interaction of the helix-turn-helix motif with DNA was the fact that the mutationof the -60 region ofpfixK did not regulatory sequences whereas phosphorylation of the receiver affect transcription activation by FixJC whereas it affected domain unmasks this motif. transcription by FixJ-PO,(Fig. 4). We havedemonstrated On the basisof DNase I and Exo I11 protection experiments, above that phosphorylation of the amino-terminal domain of we have demonstrated that FixJC protects two distinct regions FixJ allows the carboxyl-terminal domain to bind DNA. Conon the wild-type fixK promoter, one from -69 t o -44 and the ceivably, phosphorylation might also influencethe oligomerizaother overlapping the -35 region. FixJ-PO, instead protects a tion state of FixJ. single continuous region from -69 t o -31. Since the endsof the Finally our data shed some light on the mechanism of FixJfragment protected by FixJ-PO, are the same as the external mediated activationof transcription. Thecarboxyl-terminal doborders of the composite fragment protected by FixJC, it seems main of FixJ, FixJC, is shared by a variety of regulatory provery likely that both proteins recognize the same DNA se- teins including several response regulator proteins (e.g. ComA, quences. Therefore we propose that FixJ-PO, actually binds DegU, NarL, RcsB, and UhpA) and other activators that are two regions, although simultaneously, whereas FixJC can bind not in the response regulator category (e.g. GerE, LuxR, MalT, these regions independently (Fig. 5). Further support for this and SigB) (see DaRe et al. ( 8 ) for a recent update). FixJis the model comes from the protection studies at the mutant fixK third protein of this family for which it is demonstrated that promoter in which base pairs -63, -65, and -66 had been the isolated output domain can bind DNA the other examples changed. Indeed, Exo I11 protection experiments have shown are GerE,which solely consists of this domain, and a truncated that FixJ-PO, was still able to bind the -35 region of this MalT (21,22). Furthermore, FixJC and the related proteins are mutant promoter, although possibly with a weaker affinity as homologous to the so-called “region 4”located in the carboxylcompared to thewild-type promoter,even though binding to the that terminal partof u factors (7). Genetic analysis has shown (-69, -44) region was no longer detectable (Fig. 6A). Con- the helix-turn-helixmotif which is part of region 4 specifically versely, we have seen by DNase I footprinting that FixJ-PO, contacts the-35 region of bacterial promoters(23-25). Specific been demonwas able to bind the -60 region of a mutant fixK promoter binding to the-35 region of target promoters has (pFW35 in Waelkens et al. (13)) in which binding to the -35 strated so far for ComA, GerE, and MalT (21, 22, 26, 27) and, now, for FixJC and phosphorylated FixJ. Furthermore, neither region was prevented by mutation (data not shown). The composite structure of the fixK promoter, as evidenced FixJC nor phosphorylated FixJ binds to the-35 region of the which is inactive in here by biochemical means, is fully consistent with previous pFW35 mutant promoter (data not shown) genetic analysis of the fixK promoter. Firstly, atruncated fixK in vivo transcription (13). Therefore, our present working hypothesis is that FixJC, by binding to the -35 region, assists promoter with only 67 nucleotides upstream from the transcription start site is still fully active in R. meliloti (181, thus RNA polymerase in its interaction with thepromoter, may be theregion. The role of the suggesting the absenceof a FixJ-binding site upstreamof po- by replacing a70 interaction with -35 sition -67. Accordingly, we show here that the region protected (-69, -44)region, which is a region of higher affinity, would be by FixJ-PO, ends atposition -69 (Fig. 7). Secondly, Waelkens et t o facilitate the binding of FixJ-PO, to the low affinity downal. (13) have shown that FixJ-dependent promoters display a stream site. This model is supported by our observation that sequence conservation between positions -54 and -33 and that mutations preventingoccupancy of the (-69, -44)region can be mutations in this region had dramatic effects on promoter ac- restored by increasing FixJ-PO, concentration. If it thus aptivity. Here we have demonstratedthat this region corresponds pears that the (-69, -44)region has an ancillary, rather than t o a binding region for FixJ-PO,. In addition, Waelkens et al. essential, role in vitro at relatively high FixJ-PO, concentra(13) have identified a -60 region of the fixK promoter which tions, this is not true in vivo since the same mutant promoter was needed for activation by fixJ although it was absent from used in thisstudy, FW63, had only 3% activity as compared to the other FixJ-dependentpromoter, pnifA. Here we show that the wild-type promoter (13).We anticipate that this differential fact fixJ is expressed at a very low level this -60 region does interact with FixJ-PO,, or FixJC, being effect is due to the that part of the -69 to -44 region. Furthermore, transcriptionfrom in R. meliloti. In addition to protectingan extended DNA region, interacthe FW63 mutant promoter could be restored in vitro by increasing FixJ-PO, concentration (Fig. 4B)as it has also been tion of FixJ-PO, or FixJC with DNA results in the appearance of a hypersensitive siteat -62. Intriguing enough is the finding found in vivo (13). from the interactionof The difference in the protection patterns yielded by FixJC that this hypersensitive site also results and FixJ-PO, at the wild-type fixKpromoter are best explainednonphosphorylated FixJ with the promoter region (Fig. 2). A by FixJ-PO, being able t o form oligomers of higher order than likely explanation for the presence of this DNase I hypersensiFixJC. Thiscould indeed bethe case if oligomerization motifsof tive site is distortion of the DNA helix upon binding of FixJ, as even if this bindingis too weak to be detectedby DNase I and FixJ were presentin the amino-terminal regulatory domain demonstrated for other regulator proteins such as NtrC (19, Exo I11 protection experiments, resulting in the increasedac20). The FixJ oligomers would be able t o bind simultaneouslyt o cessibility of the exposed minor groove to DNase I attack (28).

FixJ DNA Binding

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