puriﬁcation and characterization of sa-lrp, a dna-binding ...erfelijkheidsleer en microbiologie,...

JOURNAL OF BACTERIOLOGY,0021-9193/00/$04.0010

July 2000, p. 3661–3672 Vol. 182, No. 13

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of Sa-Lrp, a DNA-BindingProtein from the Extreme Thermoacidophilic ArchaeonSulfolobus acidocaldarius Homologous to the Bacterial

Global Transcriptional Regulator LrpJULIUS ENORU-ETA,1 DANIEL GIGOT,2 THIA-LIN THIA-TOONG,1

NICOLAS GLANSDORFF,1,2 AND DANIEL CHARLIER1*

Erfelijkheidsleer en Microbiologie, Vrije Universiteit Brussel, and Department of Microbiology, The FlandersInteruniversity Institute for Biotechnology,1 and Laboratoire de Microbiologie, Universite Libre

de Bruxelles, and Institut de Recherches Microbiologiques J.-M. Wiame,2 B-1070 Brussels, Belgium

Received 3 February 2000/Accepted 10 April 2000

Archaea, constituting the third primary domain of life, harbor a basal transcription apparatus of theeukaryotic type, whereas curiously, a large fraction of the potential transcription regulation factors appear tobe of the bacterial type. To date, little information is available on these predicted regulators and on theintriguing interplay that necessarily has to occur with the transcription machinery. Here, we focus on Sa-lrp ofthe extremely thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, encoding an archaeal homologue ofthe Escherichia coli leucine-responsive regulatory protein Lrp, a global transcriptional regulator and genomeorganizer. Sa-lrp was shown to produce a monocistronic mRNA that was more abundant in the stationary-growth phase and produced in smaller amounts in complex medium, this down regulation being leucineindependent. We report on Sa-Lrp protein purification from S. acidocaldarius and from recombinant E. coli,both identified by N-terminal amino acid sequence determination. Recombinant Sa-Lrp was shown to behomotetrameric and to bind to its own control region; this binding proved to be leucine independent and wasstimulated at high temperatures. Interference binding experiments suggested an important role for minorgroove recognition in the Sa-Lrp–DNA complex formation, and mutant analysis indicated the importance forDNA binding of the potential helix-turn-helix motif present at the N terminus of Sa-Lrp. The DNA-bindingcapacity of purified Sa-Lrp was found to be more resistant to irreversible heat inactivation in the presence ofL-leucine, suggesting a potential physiological role of the amino acid as a cofactor.

Compared to the overwhelming amount of informationavailable on mechanisms of basal transcription and its controlin Bacteria and Eucarya, relatively little is known about thesemechanisms in Archaea, constituting the third primary domainof life (65). The crucial boxA element (now called TATA box)of archaeal promoters strongly resembles the eukaryoticTATA box of polymerase II-dependent promoters (40, 44, 45,57), the complex multisubunit composition of the archaealRNA polymerase is reminiscent of those of the eukaryotichomologues, and functional complementation between ar-chaeal and eukaryotic TATA-binding protein and transcrip-tion factor TFB (TFIIB in eukaryotes) has been demonstrated(3, 42, 43, 51, 56, 68). Therefore, the major components ofarchaeal and eukaryotic transcription initiation appear to befundamentally related. In contrast, archaeal mRNAs mostclosely resemble their bacterial homologues; they are fre-quently polycistronic and are relatively unstable, have no in-trons (except for some tRNA and rRNA genes), bear no 59 capsite, and have no or only a very short poly(A) tail. Scrutinizinggenome sequences has revealed the existence, in archaea andbacteria, of nearly identical proportions of predicted regula-tory proteins bearing a potential helix-turn-helix (HTH) DNA-binding motif, reminiscent of bacterial repressors and activa-tors; the predominant class of HTH motifs in archaea is the

winged-HTH motif (1). Therefore, Archaea appear to presentthe intriguing combination of a eukaryotic type of basic tran-scription apparatus, the activity of which would be controlledby bacterial-type regulatory proteins. This situation must haveprofound functional and evolutionary implications, and as aconsequence, studies on archaeal transcriptional regulationmay contribute not only to the deciphering of fundamentalmechanisms of transcriptional repression and activation butalso to our understanding of microbial evolution. The presentstate of knowledge calls for the urgent development of modelsystems for the study of mechanisms of specific and globaltranscriptional regulation at the molecular level, especially inextreme- and hyperthermophilic archaea. Indeed, at thepresent time, regulation of archaeal transcription initiation andmRNA stability have been addressed mostly in methanogensand halophiles, representatives of the Euryarchaeota, but verylittle information is available on extreme- and hyperthermo-philic archaea. Moreover, though mobility shift experimentsperformed with archaeal protein extracts and analyses of cis-acting regulatory elements (11, 13, 21, 22, 24, 31, 39, 46, 48, 52,53) have indicated the existence of sequence-specific DNA-binding proteins and of target sites located close to the tran-scription initiation sites of specific genes, these potential reg-ulatory elements have not been characterized thoroughly (fora recent review, see reference 33). Only one detailed study hasbeen performed (2) (see Discussion).

Previously, we reported the cloning and identification ofSa-Lrp, a thermophilic archaeal homologue of the eubacterialleucine-responsive regulatory protein Lrp (9), and recently,

* Corresponding author. Mailing address: Erfelijkheidsleer en Mi-crobiologie, Vrije Universiteit Brussel, 1-av. E. Gryson, B-1070 Brus-sels, Belgium. Phone: 32 2 526 72 79. Fax: 32 2 526 72 73. E-mail:[email protected].

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Napoli et al. (36) reported the identification of Lrs 14 fromSulfolobus solfataricus as an Lrp-like protein that binds to mul-tiple sites in its own control region and therefore might exertautoregulation. However, it is clear that S. solfataricus Lrs14and Sa-Lrp are not functionally equivalent (see Discussion). Inaddition, archaeal sequences homologous to Escherichia colilrp have been reported in Pyrococcus furiosus (16, 32) and inentirely sequenced archaeal genomes (7, 26, 27, 28, 50). How-ever, the corresponding proteins have not been studied. Bac-terial members of the Lrp/AsnC family of transcriptionalregulators are either specific transcriptional activators or re-pressors or global regulators that can exert different effects,depending on the target and the presence of a suitable cofac-tor. Which proteins among the archaeal Lrp-like proteins arespecific or global regulators is at present totally unknown. Thebest-studied member of the bacterial Lrp/AsnC family of pro-karyotic transcriptional regulators, E. coli Lrp, is a global reg-ulator that governs the expression of at least 30 genes consti-tuting the leucine/Lrp regulon. The physiological significanceis still poorly understood, but from lplacMu insertions, it wasestimated that some 50 to 75 targets might exist (8, 37, 38). Lrpfrequently superimposes its effects on more local and specificcontrols; the effect can be negative or positive and in eithercase requires leucine or is alleviated by it or is leucine inde-pendent. The latter prevails in the negative autoregulation ofthe lrp gene (63). E. coli Lrp is a small, basic, homodimericprotein that frequently binds to several targets in an array.Binding induces a pronounced bending of DNA (62), and Lrpis considered an architectural element that stimulates the for-mation of specific nucleoprotein complexes and plays a role inthe organization of the bacterial genome, in conjunction withother proteins of the histone-like type, as HU, H-NS, andintegration host factor. Mutational analysis has indicated thatthe protein is composed of three functional domains: the Nterminus involved in DNA binding, the central domain respon-sible for transcriptional activation, and the C terminus involvedin the response to leucine (41).

Our previous report was on the nucleotide sequence andtranscription initiation site of the lrp gene in the extremelyacidothermophilic crenarchaeote Sulfolobus acidocaldarius (9).Though the original report stated that the lrp gene (now des-ignated Sa-Lrp) had been cloned from an S. solfataricus DNAbank, further experimentation and amplification of the lrp geneand several other genes from S. acidocaldarius (type strainDSM 639) and S. solfataricus strains P1 and P2 (DSM 1616 andDSM 1617) with oligonucleotides based on the reported se-quence and subsequent sequence determination unambigu-ously demonstrated that the original clone was derived from S.

acidocaldarius and not from S. solfataricus. This kind of con-fusion happened to several groups and arises from incorrectspecies assignments and the distribution of mixed cultures(67).

Here we demonstrate the monocistronic nature of the Sa-lrptranscript and analyze the effects of growth phase and nutrientavailability on its synthesis. We purified and determined theN-terminal amino acid sequence of Sa-Lrp extracted from boththe original host and transgenic recombinant E. coli, charac-terized recombinant protein purified to homogeneity, studiedprotein-DNA complex formation, and analyzed the effects oftemperature and leucine (the major effector of the regulon inE. coli) on protein-DNA complex formation. We also studiedthe effects of two single-amino-acid substitutions in the poten-tial HTH motif of Sa-Lrp on its interaction with DNA. There-fore, this constitutes one of the first reports on the purificationto homogeneity and the characterization to this extent of apotential regulatory protein of thermophilic archaeal originand its interaction with DNA. The recent development of apurified in vitro transcription assay specific for S. acidocal-darius (4) should facilitate the further functional analysis ofSa-Lrp.

MATERIALS AND METHODS

Strains, media, and growth conditions. S. acidocaldarius (strain DSM 639) wasgrown aerobically at 75°C on a rotary shaker platform either in complex medium[3.1 g of KH2PO4, 2.5 g of (NH4)2SO4, 0.2 g of MgSO4 z 7H20, 0.25 g of CaCl2 z2H2O, 2.0 g of yeast extract, H2O to 1 liter and adjusted to pH 3.5 with H2SO4]or minimal medium [8.7 g of KH2PO4, 2.5 g of (NH4)2SO4, 0.2 g of MgSO4 z7H2O, 0.25 g of CaCl2 z 2H2O, H2O to 1 liter, adjusted to pH 3.5] supplementedwith 0.3% (wt/vol) glucose and 40 ml of a concentrated mineral solution per liter(20). Growth was determined from the apparent absorbance at 660 nm. Growthconditions for E. coli were described previously (19). Genotypes and descriptionsof strains and plasmids are given in Table 1. Where indicated, L-leucine (50mg/ml), kanamycin (35 mg/ml), tetracycline (15 mg/ml), and chloramphenicol (30mg/ml) were added. Isopropyl-b-D-thiogalactopyranoside (IPTG) was used at 1.0mM.

DNA preparations and manipulations. Plasmid DNA extraction was based onthe alkaline sodium dodecyl sulfate (SDS) lysis method (5) and performed withthe commercial Nucleobond AX plasmid extraction kits PC20 and PC100 (Pro-mega). Oligonucleotides were purchased from Gibco BRL and EUROGEN-TEC. Nuclease digestion, ligation, and dephosphorylation and phosphorylationof DNA fragments and oligonucleotides were performed with commercial en-zymes and buffers (Boehringer Mannheim) according to the manufacturer’sinstructions. DNA fragments obtained after endonuclease digestion or by PCRamplification were purified by agarose gel electrophoresis and recovered fromthe gel by the hot phenol extraction procedure or directly purified on the column(QIAquick PCR purification kit; Westburg). Competent cells were prepared byCaCl2 treatment (15). Enzymatic and chemical DNA sequencing was performedby the methods of Sanger et al. (47) and Maxam and Gilbert (35), respectively.

Oligonucleotide-directed mutagenesis. The single-amino-acid substitution mu-tants R44A and L48A were constructed by oligonucleotide-directed mutagenesisusing the QuickChange site-directed mutagenesis kit (Stratagene), double-

TABLE 1. Strains and plasmids used in this work

Strain Genotype Source or reference

S. acidocaldarius DSM 639 Wild-type Deutsche Sammlung vonMikroorganismen

E. coliXL1-Blue F9::Tn10 proA1BA lacIq D(lacZ)M15/recA1 endA1 gyrA96 (Nalr) thi hsdR17 (r2 m1)

supE44 relA1 lacStratagene

HMS174(DE3)/pLysS F2 recA1 hsdR(r2 m1) Rifr (DE3) pLysS (Cmr) Novagen

PlasmidspUC18 Apr, lacZ cloning vector 59pET24a Kmr, T7 polymerase-dependent expression vector NovagenpET24Lsa Sa-lrp gene cloned into NdeI-BamHI sites of pET24a This studypSPYR3 6.9-kb PstI genomic fragment of S. acidocaldarius in pKK223-3 9pOPLsa 334-bp Sa-lrp promoter region in pUC18 (BamHI-PstI) This study

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stranded pET24Lsa plasmid DNA as the template, and the complementary pairsof oligonucleotides LrpR44A-LrpR44Arev and LrpL48A-LrpL48Arev (Table 2)as the primers, according to the manufacturer’s instructions. The reaction prod-ucts were transformed into competent cells of E. coli strain XL1-Blue. PlasmidDNA extracted from individual clones was submitted to enzymatic dideoxychain-terminating sequencing (47) to verify the presence of the desired mutationand the correctness of the rest of the Sa-lrp gene.

Reverse transcriptase primer extension. S. acidocaldarius was grown at 75°C incomplex or minimal medium, supplemented with 50 mg of L-leucine per ml whenindicated, and arrested either in the exponential phase of growth (A660 of 0.4 and0.6) or in the stationary phase (A660 of 1.0). Cells were collected from 200-mlcultures by centrifugation, and total RNA was prepared by the Life Technologiesprocedure using the Trizol reagent (12). Total RNA (25 or 100 mg) was mixedwith about 40,000 cpm of 59-end 32P-labeled oligonucleotide primer (21-merSalrpRT for Sa-lrp and 22-mer SapyrBRT for pyrB) and after overnight hybrid-ization at 42°C elongated with 10 U of avian myeloblastosis virus reverse tran-scriptase (Boehringer Mannheim) at 40°C for 1 h, as described previously (9).Chain-terminating DNA sequencing reactions of the noncoding strand obtainedwith pSPYR3 plasmid DNA as the template and the same 59-end-labeled oligo-nucleotides as primer were used as reference ladders.

Reverse transcription-PCR (RT-PCR). Total RNA was extracted from a100-ml culture of S. acidocaldarius cells grown in complex medium and harvestedin the stationary phase. The RNeasy Midi kit (Qiagen) procedure was usedaccording to the manufacturer’s instructions and with inclusion of a supplemen-tary DNase I treatment to remove all traces of possible contamination withDNA. cDNA synthesis was performed with 1.0 mg of RNA, 1.0 mM each of thefour deoxynucleoside triphosphates, 1.0 mM dithiothreitol, 30 pmol of oligonu-cleotide, and 50 U of Expand Reverse Transcriptase (Boehringer Mannheim) inthe commercial buffer at 42°C for 90 min in a total volume of 20 ml, and in thepresence of 25 U of RNase inhibitor. The reaction was stopped by heating at95°C for 2 min. cDNA aliquots (3.0 ml) were used as the template in the PCRamplification step with different combinations of oligonucleotide pairs (30 pmoleach) in a total volume of 50 ml, with 0.2 mM each of the four deoxynucleosidetriphosphates and 1.5 U of PFU DNA polymerase (Promega). Initial denatur-ation was for 5 min at 94°C, PFU was added at 80°C, and synthesis was per-formed during 30 cycles (50 s at 94°C, 30 s at 50°C, and 2 min at 72°C).Elongation was allowed for an extra 10 min at 72°C after the last cycle. Samples(17 ml) were analyzed to identify the size and amount of the amplified productsby electrophoresis on a 1.5% agarose gel.

Mobility shift electrophoresis. Mobility shift experiments were performed bythe method of Fried and Crothers (17), with modifications. 32P-labeled DNAfragments, labeled at one or both 59 ends were prepared by PCR amplificationwith 59-end 32P-labeled oligonucleotides and purified by polyacrylamide gelelectrophoresis. Protein-DNA complexes were formed in 20 ml of Lrp bindingbuffer (20 mM Tris-HCl [pH 8.0], 0.4 mM EDTA, 0.1 mM dithiothreitol, 50 mMNaCl, 1 mM MgCl2, 12.5% glycerol) with 1 to 5 ng of labeled fragment and in thepresence of a 100-fold excess of nonspecific competitor (sonicated herring spermDNA) for 25 min at 37°C (unless otherwise stated) and loaded onto preelectro-phoresed 4 or 5% polyacrylamide gels in TEB buffer (89 mM Tris, 2.5 mMEDTA, 89 mM boric acid). Gels were run in the same TEB buffer at roomtemperature at 12 V/cm until penetration of the DNA into the gel and then fora further 3 h at 8 V/cm. The binding buffer is similar to the one used for E. coliLrp (63). Replacing the NaCl with 100 mM KCl or reducing the pH to 6.0, two

conditions thought to reflect more physiological conditions for Sulfolobus, didnot improve complex formation; lowering the pH even had a slight negativeeffect. The addition of bovine serum albumin (BSA) (at 50 mg/ml) had no effect.Glycerol, however, had a stabilizing effect (not shown).

DNase I footprinting. DNase I footprinting experiments with purified recom-binant Sa-Lrp protein were performed by the method of Galas and Schmitz (18)in Lrp binding buffer (see above) as described by Charlier et al. (10).

Sa-Lrp protein purification. Recombinant Sa-Lrp protein was purified from a2-liter culture of E. coli strain HMS174(DE3)pLysS carrying plasmid pET24Lsa,grown in complex medium supplemented with chloramphenicol and kanamycinand induced with 1.0 mM IPTG at a cell density of 6 3 108/ml for 5 h. Cells werecollected by centrifugation, rinsed with extraction buffer (50 mM Tris-HCl [pH8.0]), resuspended in 15 ml of extraction buffer, and disrupted by sonication in aRaytheon sonicator (250 W) for 25 min at 4°C. Cell debris were removed bycentrifugation for 30 min at 30,000 3 g. The pellet was discarded, and thesupernatant was incubated at 70°C for 5 min, with shaking. An extra 4 ml ofextraction buffer was added, and the denatured proteins were removed by cen-trifugation for 30 min at 30,000 3 g. The supernatant was loaded onto a Mono-SHR 10/10 ion exchange column (fast protein liquid chromatography; Pharmacia)preequilibrated with extraction buffer, and eluted with a NaCl gradient (0 to 1.0M). Fractions containing Sa-Lrp protein (identified by mobility shift electro-phoresis) were pooled, concentrated about fourfold on Centricon 10 membranefilters (Amicon), and further purified by gel filtration chromatography on aSuperose P12 HR 10/30 column, equilibrated with extraction buffer containing0.1 M NaCl. Sa-Lrp emerged as a single peak with an apparent molecular massof 67.0 kDa. Sa-Lrp-containing fractions were pooled, concentrated, and de-salted on Centricon filters. The final yield was about 10 mg of Sa-Lrp proteinpurified to electrophoretic homogeneity. The R45A and L48A mutant Sa-Lrpproteins were purified by the same procedure. As these proteins are severelyaffected in the DNA-binding capacity, the mobility shift assay could not be usedto monitor the protein in the chromotographic steps, and the purification strat-egy relied entirely on the characteristic behavior of the wild-type protein (seealso purification from the native host).

To establish a protocol for purification of Sa-Lrp protein from the native hostS. acidocaldarius, we took advantage of the properties of the recombinant pro-tein extracted from E. coli. Cells from a 2-liter culture of S. acidocaldarius strainDSM 639 grown in complex medium and arrested in the stationary phase wereharvested by centrifugation at 4°C, resuspended in 15 ml of extraction buffer, anddisrupted by sonication in a Raytheon sonicator at 250 W for 30 min at 4°C. Celldebris was removed by centrifugation for 30 min at 45,000 3 g. The cell-freesupernatant was submitted to an ammonium sulfate precipitation step at 50%saturation and centrifuged for 60 min at 30,000 3 g. The pellet was discarded,and the supernatant was precipitated at 85% saturation in ammonium sulfate.Precipitated proteins were collected by centrifugation for 20 min at 10,000 3 g.The pellet was dissolved in 5 ml of extraction buffer and dialyzed overnightagainst 1 liter of extraction buffer. The dialyzed protein solution was loaded ontoa Mono-S HR 10/10 column (fast protein liquid chromatography; Pharmacia)equilibrated with extraction buffer and eluted with a NaCl gradient (0 to 1.0 M).Fractions containing Sa-Lrp protein (based on the elution profile of the recom-binant protein and SDS-polyacrylamide gel electrophoresis [SDS-PAGE]) werepooled and concentrated ca. eightfold on a Centricon 10 membrane filter andfurther purified by gel filtration chromatography on a Superose P12 HR 10/30column equilibrated with extraction buffer containing 0.1 M NaCl. Fractions

TABLE 2. Oligonucleotides used in this work

Oligonucleotide Sequence Use

SalrpRT 59-CTTCTGCTAATCTTCTGAGAC-39 RT primer extension of Sa-lrpSapyrBRT 59-CTGTTAGGGCAAATATGTCCTC-39 RT primer extension of pyrB1 59-CTAGGATCTTCCTTTACCAC-39 RT-PCR of Sa-lrp2 59-GAAAAAAGATAGAAATAGACGC-39 RT-PCR of Sa-lrp3 59-CCATATATTGATGTAAAGCG-39 RT-PCR of Sa-lrp4 59-GTTCAACTTATACTATCCGG-39 RT-PCR of Sa-lrp5 59-GGTGAATATGATGTCATGC-39 RT-PCR of Sa-lrpSalrpOP1 59-AACTGCAGGTTTTTTATCTATTGCGTC-39 Amplification of Sa-lrp promoter-operatorSalrpOP2 59-CGGGATCCGACTGCGCTCATAAGTTTATCAGTGG-39 Amplification of Sa-lrp promoter-operatorEclrpOP1 59-AACTGCAGCTCTGCTACTTAAATTTCCCGC-39 Amplification of E. coli lrp promoter-operatorEclrpOP2 59-CGGGATCCGGTTATGCGCATATAAGAATACTG-39 Amplification of E. coli lrp promoter-operatorPflrpOP1 59-AACTGCAGGGATGGGTCAAGCACTGATTCC-39 Amplification of P. furiosus lrp promoter-operatorPflrpOP2 59-CGGGATCCCTCAGTGAAGGGCGTTCTC-39 Amplification of P. furiosus lrp promoter-operatorPfgdhOP1 59-CGGGATCCAAGCTTTATATAGGCTATTGC-39 Amplification of P. furiosus gdh promoter-operatorPfgdhOP2 59-GGAATTCGAACTCAAGAGCTTCTTCAC-39 Amplification of P. furiosus gdh promoter-operatorLrpR44A 59-CGAACACTACATAATGCACTTATGAGGCTAG-39 Sa-Lrp R44A mutant constructionLrpR44Arev 59-CTAGCCTCATAAGTGCATTATGTAGTGTTCG-39 Sa-Lrp R44A mutant constructionLrpL48A 59-GACTTATGAGGGCAGTCCAAGAAGG-39 Sa-Lrp L48A mutant constructionLrpL48Arev 59-CCTTCTTGGACTGCCCTCATAAGTC-39 Sa-Lrp L48A mutant construction

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containing Sa-Lrp (peak near 67.0 kDa and analysis by SDS-PAGE) were pooledand concentrated on a Centricon 10 membrane filter to a volume of 200 ml.When subjected to SDS-PAGE, this material showed two bands. The bandcorresponding to 16.4 kDa was recovered and subjected to N-terminal aminoacid microsequencing and was thus proved to correspond to Sa-Lrp. It wasimpossible to establish unambiguously the N-terminal amino acid sequence ofthe major contaminant corresponding to a subunit molecular mass of 10.0 kDa,most likely because the band on the SDS-polyacrylamide gel corresponds tomore than one protein.

RESULTS

Modulation of monocistronic lrp mRNA levels in S. acido-caldarius. Previously we have shown that in S. acidocaldariuscells grown in complex medium, Sa-lrp transcription is drivenby a strong and typical archaeal promoter initiated at an Aresidue located 8 nucleotides (nt) upstream of the ATG initi-ator codon (9). To determine the effects of nutrients andgrowth phase on the abundance of Sa-lrp transcription, weperformed quantitative primer extension experiments with to-tal RNA extracted from S. acidocaldarius cells grown on com-plex medium or on minimal medium either devoid of or sup-plemented with L-leucine and harvested either in theexponential phase of growth, near the end of the exponentialphase, or in the stationary phase (see Materials and Methods).The results (Fig. 1a) indicate that Sa-lrp transcription is aboutthreefold more abundant in the stationary phase than in theexponential phase (compare lanes 1 and 3); this effect is spe-cific, since it was not observed with the same RNA prepara-tions for mRNAs of pyrimidine biosynthetic genes (Fig. 1b).Transcription of the Sa-lrp gene was repressed approximatelytwofold in complex medium (Fig. 1a, compare lanes 1 and 4)but leucine had no detectable effect (lanes 4 and 5). In allinstances, Sa-lrp transcription was initiated only at the same,previously identified site (9), indicating that under all condi-tions examined, transcription was initiated from a single pro-moter. This was confirmed by RT-PCR experiments whichdemonstrated, moreover, that the Sa-lrp messenger is mono-cistronic. Indeed, whereas a strong amplified signal was ob-tained in the RT-PCR performed with a pair of oligonucleo-tides (oligonucleotides 1 and 2 [Fig. 1c and d]) correspondingto the 59 and 39 ends of the Sa-lrp open reading frame (ORF),no or only a very weak signal was detected when one of theoligonucleotides constituting the pairs was located in orf4 (oli-gonucleotide 4), preceding the Sa-lrp gene, or downstream of it(oligonucleotide 3), respectively. The extremely weak signalmeasured with the combination of oligonucleotides 1 and 4(Fig. 1d, lanes 3 and 4) indicates the quasi absence (0.6%readthrough) of Sa-lrp mRNA produced by readthrough tran-scription initiated from pyrimidine gene promoters locatedupstream (D. Charlier, T.-L. Thia-Toong, V. Durbecq, M.Roovers, and N. Glansdorff, Abstr. 26th FEBS Meet., abstr.s354, 1999). Similarly, the relatively small amount of cDNAsynthesized with oligonucleotide 3 as the primer indicates thatthe majority of the transcripts do not proceed beyond theSa-lrp gene and most likely transcription stops at the potentialtype I transcriptional stop signal (TTTTTATT), located 1 ntdownstream of the TAA stop codon (see Discussion also). Thedensitometric analysis indicated 3.5 and 5.3% readthrough, asmeasured by amplification of the cDNA with oligonucleotides2 and 5, respectively.

Overexpression of Sa-lrp in E. coli and purification andcharacterization of the recombinant protein (Sa-Lrp). The155-amino-acid potential coding region of Sa-lrp, including thestop codon, was amplified by PCR using plasmid pSPYR3bearing a 6.9-kb genomic PstI fragment as a template (9) andligated into NdeI- and BamHI-digested expression vectorpET24a, giving rise to pET24Lsa. In this construct, Sa-lrp is

expressed from a T7 RNA polymerase-dependent and LacI-repressible promoter. A band not present in the control andcorresponding to a 16.2-kDa subunit was detected by SDS-PAGE in cell extracts of IPTG-induced cells bearing this re-combinant pET24Lsa plasmid; this value is compatible withthe calculated molecular mass of 17,640 daltons deduced fromthe DNA sequence of Sa-lrp (with omission of the initiatormethionine residue [see below]). Recombinant Sa-Lrp was pu-rified to electrophoretic homogeneity (Fig. 2a) from a 2-literIPTG-induced culture by a combination of three steps: heattreatment of the cell extract, Mono-S ion exchange chroma-tography, and gel filtration on a Superose P12 HR 10/30 col-umn (for details, see Materials and Methods). The presence ofSa-Lrp protein in the different fractions was assayed by mobil-ity shift electrophoresis, based on the capacity of Sa-Lrp tobind to its own promoter-operator region (see below). On themolecular sieve column, Sa-Lrp emerged as a peak with anapparent molecular mass of 67.0 kDa (Fig. 2b). To confirm thisvalue, a mixture of purified Sa-Lrp and BSA (67.0 kDa) wassubjected to gel filtration chromatography; the two proteinswere unseparable and eluted as a single symmetric peak. Incontrast, a mixture of Sa-Lrp and ovalbumin (43.0 kDa) elutedas two separate peaks, Sa-Lrp eluting first (not shown). Whensubjected to SDS-PAGE, purified recombinant Sa-Lrp mi-grated as a single 16.2-kDa band (Fig. 2a, lane 5). Combined,these data are most consistent with the native recombinantprotein being a homotetramer. The identity of the purifiedprotein was confirmed by determining the N-terminal aminoacid sequence of 13 residues (SDRKKIEIDAIDK), which per-fectly matches the amino acid sequence deduced from theDNA sequence of the predicted ORF but lacks the initiatormethionine. Methionine removal by E. coli methionyl-amino-peptidase (MAP) depends mainly on the nature of the secondamino acid residue in the polypeptide chain; the catalytic ef-ficiency of MAP decreases with increasing length of the sidechain (23). The second residue in the Sa-Lrp protein is serine;the efficient maturation of the recombinant protein is thereforein good agreement with the proposed rule, and moreover,serine is one of the most abundant N-terminal amino acidsfound among cytosolic proteins in E. coli. The same situationprevails for Sa-Lrp protein synthesized in the original host.Sa-Lrp purified from S. acidocaldarius (see Materials andMethods) behaved as a homotetrameric protein of 16.4-kDasubunits, as judged by gel filtration and SDS-PAGE. N-termi-nal amino acid sequencing of eight residues (SDRKKIEI) un-ambiguously confirmed the identity of the purified protein,identified its translational start, and indicated absence of theinitiator methionine from the mature protein, also in the orig-inal archaeal host. Therefore, the purified recombinant proteinutilized in further in vitro work has exactly the same amino acidsequence as the protein present in the original host (unlessposttranslational modification of Sa-Lrp occurs in S. acidocal-darius).

In vitro DNA binding of Sa-Lrp. The DNA-binding capacityand specificity of purified recombinant Sa-Lrp were investi-gated by mobility shift electrophoresis. In the presence of alarge excess (.100-fold) of nonspecific competitor (sonicatedherring sperm DNA), Sa-Lrp bound to a 334-bp DNA frag-ment (positions 2278 to 156) encompassing the promoter andtranscription initiation site of Sa-lrp. Even at the lowest Sa-Lrpconcentration (about 140 nM) at which we could detect com-plex formation under these conditions, Sa-Lrp–DNA com-plexes hardly penetrated the 4 and 5% polyacrylamide gels(Fig. 3a). The apparent dissociation constant Kd for Sa-Lrpbinding to its own control region, as determined from thehalf-saturation point in mobility shift experiments conducted



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at low target DNA concentrations and at 37°C, is about 200 nM(mean value from several experiments). The addition of L-leucine, at various concentrations and up to 30 mM, in thebinding assay and in the gel solution and the running buffer,did not significantly affect Lrp binding (Fig. 3a).

Binding to subfragments of the 334-bp operator fragmentwas performed with partially RsaI-digested operator DNA la-beled at both 59 ends, resulting in a mixture of three fragments,the intact 334-bp operator fragment and subfragments of 204bp (2147 to 156) and 130 bp (2278 to 2147) (Fig. 3b). Themobility shift experiment performed with this mixture showedthat Sa-Lrp bound to the intact fragment with an approxi-mately fourfold-higher affinity than to the 204-bp subfragment,whereas binding to the 130-bp fragment was nearly undetect-able, as judged from the decrease of free DNA bands (Fig. 3b).Similarly, Sa-Lrp was shown to bind better to the intact 334-bpfragment than to the subfragments of 218 bp (2278 to 261)and 116 bp (261 to 156) generated by DraI digestion (Fig. 3c).Sa-Lrp binding to the E. coli and P. furiosus lrp control regionscould also be detected, though with an approximately three-and fivefold-lower apparent affinity, respectively (Fig. 3d ande), and binding to the P. furiosus gdh promoter (16) was stillweaker (Fig. 3f). Sa-Lrp appears therefore to bind DNA withbut relatively weak sequence specificity.

Effect of a single-amino-acid substitution in the potentialHTH motif of Sa-Lrp on DNA binding. In its N terminus,Sa-Lrp bears a stretch that might fulfill the major requirementsof a potential HTH motif (9). Arginine 44 and leucine 48 ofSa-Lrp (starting from serine as position 1 of the mature pro-tein) are highly conserved among bacterial members of theLrp/AsnC family and related archaeal Lrp-like proteins. Byoligonucleotide-directed mutagenesis, we have replaced Arg44 and Leu 48 of Sa-Lrp with alanine (see Materials andMethods). Mobility shift experiments performed with freshlypurified wild-type and mutant proteins and with the 334-bpSa-lrp control region as the target demonstrated that bothsubstitutions severely impaired DNA binding; no complex for-mation was detected even at mutant protein concentrations20-fold higher than the one required to observe 50% bindingwith wild-type Sa-Lrp (Fig. 4).

Effect of small, groove-specific DNA ligands on Sa-Lrp bind-ing. Distamycin is a basic oligopeptide of which the threeN-methylpyrrole rings interact noncovalently with the nar-rower minor groove of A1T-rich sequences containing clustersof at least four A2T pairs (14, 58). The addition of increasingconcentrations of distamycin in the Sa-Lrp binding assay re-sulted in a gradual decrease of complex formation, as deter-mined by mobility shift electrophoresis (Fig. 5a). A significantinterference effect was already observed at 5 mM distamycin,

FIG. 1. (a) Quantitative determination of Sa-lrp transcripts and mapping ofthe transcription start site by primer extension. Lanes 1 to 3, Sa-lrp primerextension reactions with 25 mg of total RNA extracted from S. acidocaldariuscells grown in complex medium and arrested in the exponential (exp.) phase, atthe end of the exponential phase, and in the stationary (stat.) phase, respectively;lanes 4 and 5, Sa-lrp primer extension reaction mixtures with 25 mg of total RNAof cells grown in minimal medium (min) devoid of and supplemented with 50 mg

of L-leucine (leu) per ml, respectively, and arrested in the exponential phase;lanes G, A, T, and C, chain-terminating DNA sequencing reactions of thenoncoding strand obtained with the same 32P-labeled oligonucleotide used toperform the extension reactions. (b) Primer extension reactions with the sameRNA preparations as in panel a but with 100 mg of total RNA and a pyrBoligonucleotide as the primer. Exposure time was threefold longer than forSa-lrp. (c) Schematic presentation of the Sa-lrp region. 11 indicates the tran-scription start site. A small vertical bar indicates the position of the translationalstop codon. The positions of oligonucleotides used as primers in the RT-PCRsand their polarity are indicated by small arrows. (d) Analysis by agarose gelelectrophoresis (1.5%) of reverse transcription-PCR performed with S. acido-caldarius RNA, Expand reverse transcriptase, Pfu DNA polymerase, and differ-ent combinations of oligonucleotide (oligo) pairs. Primers 1 and 3 were used tosynthesize cDNA, which was then used as the template and amplified in a secondPCR using oligonucleotide 2, 4, or 5. Lanes 2, 4, 6, and 8 are negative-controlreactions performed in the absence (2) of reverse transcriptase (RT) to detectpossible traces of DNA contamination in the RNA preparation.



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and binding was totally abolished at 250 mM. Interestingly, aremarkable effect on migration was also observed for freeDNA, indicating extensive binding of distamycin to the Sa-lrpcontrol region and local structural deformation of the doublehelix. A similar effect of distamycin on the migration of nakedDNA was observed with the region upstream of the P1 pro-

moter of the E. coli carAB operon (D. Charlier, unpublisheddata) and the E. coli pap operon (66). Methyl green binds tohydrophobic surfaces in the major groove. Although this smallligand also affects Sa-Lrp binding, the effect was clearly lesspronounced, requiring about 25-fold-higher concentrations of

FIG. 2. (a) Determination of the molecular mass of the recombinant Sa-Lrpsubunit and degree of purity of the Sa-Lrp preparation by SDS-PAGE analysison a 4 to 20% gradient gel. Lanes 1 to 3, 10, 5.0, and 2.5 ml, respectively, of crudeextract from a 2-liter culture of E. coli strain HMS174(DE3)/pLysS/pET24Lsainduced with 1 mM IPTG for 5 h; lane 4, 5.0 ml of supernatant of the samerecombinant E. coli extract after thermodenaturation of host cell proteins at70°C for 5 min; lane 5, 6.0 mg of purified Sa-Lrp protein; lane 6, molecular massstandards. The arrow indicates the position of the Sa-Lrp subunit. Protein bandswere visualized by staining with Coomassie brilliant blue. (b) Elution profile ofprotein fractions containing recombinant Sa-Lrp (identified by DNA-bindingassay) obtained by MonoS ion exchange column chromatography, pooled, con-centrated, and charged onto a Superose P12 HR 10/30 column. The arrowindicates the position of the Sa-Lrp protein (identified by DNA binding andN-terminal amino acid sequence determination). The column was calibratedusing aldolase (158 kDa), BSA (67 kDa), ovalbumin (43 kDa), chymotrypsinogenA (25 kDa), and RNase A (13.7 kDa).

FIG. 3. Detection of Sa-Lrp binding to various potential target sites by mo-bility shift electrophoresis on 5% polyacrylamide gels to separate free DNA (F)from Sa-Lrp-bound (B) DNA molecules. The concentrations of pure wild-typerecombinant Sa-Lrp (in micrograms per milliliter) are indicated above the lanes.(a) Binding to the 334-bp Sa-lrp promoter-operator fragment (2278 to 156) inthe absence of L-leucine and in the presence of 30 mM L-leucine in the bindingbuffer, the electrophoresis running buffer, and the gel solution, as indicated. (bto f) Binding to a partial RsaI digest of the 334-bp fragment (b), to a full DraIdigest of the 334-bp fragment (c), to the E. coli lrp promoter region (d), to theP. furiosus lrp promoter region (e), and to the P. furiosus gdh promoter region (f).



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methyl green than of distamycin to obtain a comparable degreeof binding interference (Fig. 5b). Since the DNA-binding af-finities of these two ligands have been claimed to be apparentlyequivalent (29), these results emphasize the importance ofminor groove geometry for Sa-Lrp binding. Previously we havereported a similar interference effect of both ligands with bind-ing of the E. coli arginine repressor (61), a member of thewinged HTH family of DNA-binding proteins (54) that makecontacts to minor and major groove determinants of the op-erators.

DNase I footprinting of Sa-Lrp. DNase I footprinting ofSa-Lrp protein to a 334-bp DNA fragment bearing the Sa-lrpcontrol region revealed on each strand a very large and not yetwell delimited region of interaction covering more than 150 nt,overlapping the TATA box and the transcription initiation site.This large zone of apparent protein-DNA contact can be sub-divided into several smaller regions of Sa-Lrp-induced protec-tion against nuclease attack separated from each other by shortregions (a few nucleotides long) of normal accessibility toDNase I (Fig. 6). Moreover, a few sites hypersensitive toDNase I cleavage were created upon Sa-Lrp binding, whereasthe region covering the TATA box was rather resistant toDNase I action, even in the absence of Sa-Lrp. It is well-knownthat the minor groove of AT-rich sequences is narrow, hencerestricting the accessibility to the nuclease. The extent and thecomplexity of the Sa-Lrp footprint most likely reflect the in-teraction of more than one protein molecule with the Sa-lrpcontrol region and the formation of a higher-order structurepossibly involving DNA bending, looping, and/or wrapping.Further experimentation is required to analyze the details ofthis complex protein-DNA interaction.

Sa-Lrp has an intrinsically thermostable DNA-binding ac-tivity. The functional thermostability of Sa-Lrp was deter-mined by incubating aliquots of purified recombinant proteinat 0.6 mg/ml for 15 min at various temperatures from 75 to100°C in 5°C increments and a subsequent assay of the residualbinding capacity to the 334-bp Sa-lrp promoter-operator frag-ment in a mobility shift electrophoresis experiment. Up to80°C, no irreversible inactivation could be observed, whereasupon incubation of the protein at 85°C, the residual bindingcapacity started to decline progressively, and at 100°C, thebinding capacity was completely and irreversibly abolished

(Fig. 7a). Interestingly, the addition of L-leucine at 10 mM tothe protein solution prior to incubation at a high temperature(92°C) stabilized the protein against heat inactivation (Fig. 7b).A similar but less pronounced effect could be observed withL-valine, whereas L-alanine had no significant effect. Therefore,the stabilizing effect of leucine appears to be specific and issuggestive of a physiologically relevant interaction of thisamino acid with Sa-Lrp protein.

Sa-Lrp–operator complex formation is stimulated at hightemperatures. To determine the effects of temperature oncomplex formation and complex stability, identical amounts ofpurified recombinant Sa-Lrp were incubated in the presence ofend-labeled 334-bp operator DNA for 15 min at 37°C, thenshifted to various temperatures, incubated for a further 15 min,and immediately loaded on a polyacrylamide gel to separateprotein-DNA complexes from free DNA molecules (Fig. 8).The results indicated an increase in complex formation whenthe temperature was raised from 37 to 60°C, and this effect waseven more pronounced at 70 and 80°C. From 90°C on, a de-crease could be observed, with complex formation at 90°C stillhigher than that measured at 37°C. At 95°C, some bindingcould still be detected, but a large proportion of the double-stranded target DNA molecules were denatured; at 100°C,complex formation had nearly vanished due to inactivation ofthe protein and denaturation of the target DNA molecules.The labeled material migrating with a velocity intermediate

FIG. 4. Analysis of wild-type and R44A and L48A substituted Sa-Lrp proteinbinding to the labeled 334-bp Sa-lrp promoter-operator fragment by mobilityshift electrophoresis to separate free DNA (F) from protein-DNA complexes(B). The protein concentrations used in the different wells are indicated (inmicrograms per milliliter) above the lanes. The wild-type protein sample used inthis experiment was different from the one used in all other experiments de-scribed and was prepared freshly, in parallel with the R44A and L48A substitutedproteins. All protein preparations exhibited a similar degree of purity.

FIG. 5. Interference effect on Sa-Lrp binding by distamycin A and methylgreen. End-labeled 334-bp Sa-lrp promoter-operator DNA was incubated at 37°Cwith 80 mg of Sa-Lrp per ml and increasing concentrations of small, groove-specific ligand. Sa-Lrp-bound DNA molecules (B) were separated from freeDNA (F) by mobility shift electrophoresis on 5% polyacrylamide gels. Lanes 1,DNA only; lanes 2, without small ligand; lanes 3 to 10, with 0.5, 1.25, 2.5, 3.75,5.0, 25.0, 50, and 250 mM distamycin (a) or methyl green (b).



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between those of protein-DNA complexes and free single-stranded DNA observed at very high temperatures (lanes 7 and8) might represent Sa-Lrp-bound single-stranded DNA mole-cules or, more likely, partially denatured double-stranded mol-ecules stabilized by bound Sa-Lrp against heat denaturation.Combined, these experiments clearly demonstrate that Sa-Lrpis an intrinsically thermostable DNA-binding protein and thatits interaction with the DNA is stimulated at high temperaturesand physiological temperatures.

DISCUSSION

In order to understand archaeal molecular physiology andthe molecular mechanisms modulating archaeal gene expres-sion, we took advantage of the recently cloned Sa-lrp gene of S.acidocaldarius (9) (also see the introduction), encoding a ho-mologue of the E. coli leucine-responsive regulatory proteinLrp, a global transcriptional regulator and genome organizer,to start investigations on this potential archaeal transcriptionalregulator (Sa-Lrp) and its interaction with DNA. Althoughgenerally more than one hypothetical regulatory protein be-longing to the Lrp/AsnC family of DNA-binding proteins canbe recognized in bacterial and archaeal genomes (up to sevenin B. subtilis [30]), most of which must be local and specificrather than global regulators, we may have identified a homo-logue of the global bacterial regulator that might fulfill a sim-ilar global function in an archaeon. Without physiological data,which are currently very difficult to gather for thermophilicarchaea, this is difficult to prove. Nevertheless, a comparison ofSa-Lrp with the S. solfataricus (type strain P2) genome hasrevealed the existence of a homologue (c08 044) that shows74% amino acid sequence identity with Sa-Lrp; moreover, thecorresponding genes are located in an identical genomic envi-

FIG. 6. (a) Part of an autoradiogram of a DNase I footprinting experiment ofthe 334-bp Sa-lrp promoter-operator region (lower strand labeled) protectedwith Sa-Lrp. A1G and C1T are the corresponding Maxam-Gilbert sequencingladders. The DNase I footprinting experiment was done in the absence of Sa-Lrp(0) and with increasing concentrations of Sa-Lrp. The global region of interac-tion is boxed. Positions that remain accessible to DNase I in the presence ofSa-Lrp (small black circles) and hyperreactive sites for DNase I cleavage in thepresence of Sa-Lrp (horizontal black lines) are indicated. (b) Nucleotide se-quence of the 334-bp fragment bearing the Sa-lrp promoter-operator region. 11indicates the transcription start site, the TATA box is underlined, and an asteriskmarks the translational ATG initiator codon. The global region that by DNase Ifootprinting appears to interact with Sa-Lrp is shaded. Sites that remain acces-sible to DNase I in the presence of Sa-Lrp (small black circles) and sites thatbecome hypersensitive to DNase I cleavage in the presence of Sa-Lrp (shortvertical arrows) are indicated.



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ronment. Since this ORF is the one among all potential S.solfataricus protein sequences that gives the best score with E.coli Lrp (30% amino acid sequence identity), Sa-Lrp and itshomologue from S. solfataricus are the best candidates to fulfilla task similar to that of bacterial Lrp in the Sulfolobales. More-over, several other lines of evidence are at least compatiblewith this proposal. (i) There is the relative abundance of Sa-lrpmRNA and protein synthesis, especially for a regulatory pro-tein (difficult to quantify exactly, but in any case the Sa-lrpmessenger is at least 10-fold-more abundant than the pyrimi-dine biosynthetic gene transcripts). (ii) The modulation of Sa-lrp transcription as affected by growth phase and nutrient avail-ability is reminiscent of E. coli lrp transcription. (iii) Like E.coli Lrp, Sa-Lrp binds to its own control region in a leucine-independent manner, suggestive of leucine-independent auto-regulation. (iv) Most significantly, L-leucine specifically pro-tects the archaeal Sa-Lrp binding capacity against irreversibleheat inactivation; this effect likely reflects a physiologicallysignificant interaction of leucine with the Sa-Lrp protein andsuggests that leucine might function as the main effector of ahypothetical regulon. In addition, the high predicted (and ex-perimentally confirmed [data not shown]) pI of 8.9 for the S.acidocaldarius protein (9.0 for the S. solfataricus homologue) ischaracteristic for Lrp proteins, and the subunit length and theabsence of tryptophan residues add to the similarity of thebacterial and archaeal Lrp-like proteins. The specific transcrip-tional regulators of the Lrp/AsnC family appear to be some-what different. Pseudomonas putida BkdR, a specific transcrip-tional activator of the bkd operon has a lower pI of 5.89 (34),and the pI of E. coli AsnC, a transcriptional activator of theLrp-like family is 6.35.

Sa-Lrp is a tetrameric protein, whereas E. coli Lrp is adimer; this higher oligomeric form of the regulatory proteinobserved in the extreme thermophilic archaeon might be re-lated to its thermal stability. Indeed, organization into a higheroligomeric form is one of the strategies that nature has devel-oped for stabilizing the native conformation of proteins at hightemperatures, as clearly demonstrated for dodecameric orni-thine carbamoyltransferase of P. furiosus (60). However, itshould be noted that other mesophilic and more distantly re-lated members of the AsnC/Lrp family like Bacillus subtilisLrpC and P. putida BkdR are tetramers as well (55).

In all growth conditions tested (exponential and stationaryphase, complex and minimal medium, also supplemented withleucine), Sa-lrp transcription was initiated with an A residue 8nt upstream of the translational start. Sequence analysis of theupstream region is consistent with the relative abundance ofthe Sa-lrp messenger. The Sa-lrp promoter bears typical ar-chaeal TATA promoter (TTTAAC) and upstream BRE (tran-scription factor B recognition element) elements that show agood match to the consensus sequence and are ideally situatedwith respect to the transcription start site. We have demon-strated that the Sa-lrp transcript is monocistronic and mostlikely stops at the pyrimidine-rich stretch (TTTTTATT) lo-cated 1 nt downstream of the translational stop codon. Tran-scriptional terminator sequences are not yet well defined inarchaea but have been proposed to be of two types in S.solfataricus. Type I terminators (45) consist of a pyrimidine

FIG. 7. (a) Effect of heat on the binding capacity of Sa-Lrp. Aliquots ofpurified Sa-Lrp protein (at 0.6 mg/ml) were incubated for 15 min at varioustemperatures, chilled on ice, centrifuged for 5 min in an Eppendorf centrifuge,and stored on ice before incubation of identical volumes (containing 2.0 mg ofinitially active protein) with the 59-end 32P-labeled 334-bp Sa-lrp promoter-operator fragment at 37°C. The experiment was done without Sa-Lrp (lane 1),with 2.0 mg of nontreated Sa-Lrp (lane 2), and with Sa-Lrp treated at 75, 80, 85,90, 95, and 100°C (lanes 3 to 8). The positions of free DNA (F) and Sa-Lrp-bound DNA molecules (B) are indicated. (b) Effects of leucine, valine, and

alanine on heat inactivation of Sa-Lrp. Aliquots of purified protein (at 0.6 mg/ml)were incubated in the presence of 10 mM of L-valine, or L-alanine at 92°C for 20min and further treated and used in a mobility shift assay as in panel 2. Theexperiment was done without Sa-Lrp (lane 1), with 2.0 mg of nontreated Sa-Lrp(lane 2), and with 2.0 mg of Sa-Lrp treated at 92°C in the presence of leucine,valine, or alanine and in the absence of any amino acid (a.a.) (lanes 3 to 6).



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stretch similar to the one proposed here for Sa-lrp. The appar-ently more frequently occurring type II terminators correspondto the W2TGTATN2W2 consensus sequence (W 5 A or T)followed by a stem-loop structure 11 to 81 nt downstream (49).Another potential transcriptional type I stop signal (TTTTTT)precedes the Sa-lrp transcription inition site by 21 nt and over-laps the TATA box (TTTAAC) by 3 nt. This sequence appearsto constitute the very efficient terminator signal for one wing ofa bipolar pyrimidine operon that precedes Sa-lrp on the ge-nome and is transcribed in the same direction (Charlier et al.,Abstr. 26th FEBS Meet., 1999).

A ribosome binding site appears to be absent from the shortSa-lrp leader preceding the ATG codon, and one could not berecognized downstream of the translational initiation codon.This observation raises the possibility that Sa-lrp messenger istranslated in the absence of a conventional ribosome bindingsite. Apparently, this situation does not constitute a severehandicap for efficient translation; a similar situation has beenreported for bacterial, archaeal, and eucaryal genes, even forproteins that are produced in large amounts (25).

We have demonstrated binding of the 67.0-kDa homotet-rameric Sa-Lrp protein to its own control region; moreover,this interaction was shown to be leucine independent. Mobilityshift electrophoresis, DNase I footprinting, and small ligandbinding interference experiments indicated that a large DNAregion overlapping the promoter elements is involved in Sa-Lrp–DNA interaction and that the minor groove of the DNAhelix is particularly important for complex formation. Negativeautoregulation of E. coli lrp is also independent of leucine andinvolves the binding of several protein molecules over a dis-tance covering more than 200 bp.

The stimulation of Sa-Lrp–DNA complex formation ob-

served at high temperatures, up to 80°C at least, emphasizesthe thermophilic character of this interaction. In conjunctionwith the intrinsic thermostability of the protein, its increasedfunctional thermotolerance observed in the presence ofleucine (suggestive of a specific interaction of Sa-Lrp with thisamino acid [the major effector of the E. coli Lrp regulon]), andthe abundance, these observations suggest that Sa-Lrp mayfulfill the role of a global transcriptional regulator of a hypo-thetic regulon in this extremely thermophilic crenarchaeote.

E. coli Lrp bears in its N-terminal part a potential HTHmotif (66) that from a mutational analysis has been proposedto be responsible for binding to DNA (41). The equivalentregion in the Sulfolobus protein might very well adopt a similarfold, as most of the major requirements of a HTH motif (6) arefulfilled (discussed reference 9). The severe reduction in DNA-binding capacity observed in the single-amino-acid substitu-tions R44A and L48A located in this region lends furthersupport to the existence of this motif and its importance forDNA binding. There is as yet no structural model available forany of the bacterial or archaeal members of the Lrp-like familyof DNA-binding regulatory proteins. A more detailed inter-pretation of the observed effects is therefore premature, andunfortunately, the lack of well-developed molecular tools forthermophilic archaea strongly limits the functional analysis invivo. However, the present evidence gathered on Sa-Lrp issufficient to warrant further studies on this DNA-binding pro-tein. Attempts to grow crystals for the structure determinationby X-ray diffraction are in progress (in collaboration with D.Maes, Ultrastructure Department, Vrije Universiteit Brussel,Brussels, Belgium), and in vitro transcription assays will beperformed to gather further evidence for a physiologically rel-evant regulatory function for the archaeal regulator (in collab-oration with S. Bell and S. Jackson, Cambridge University,Cambridge, United Kingdom).

Recently, Napoli et al. (36) presented the Lrs 14 protein ofS. solfataricus as an Lrp-like protein that binds to its owncontrol region. Lrs 14 and Sa-Lrp share only 10.3% amino acidsequence identity; this rather weak similarity and sequencecomparisons bringing to light the existence of a real homo-logue (see above) make it very unlikely that Lrs 14 and Sa-Lrpwould be functional equivalents. Though both proteins show agrowth stage- and nutrient composition-dependent synthesisthat is reminiscent of that of E. coli Lrp, the pIs of the twoproteins, 8.9 and 8.2 for the S. acidocaldarius and solfataricusproteins, respectively (9.1 for E. coli Lrp), are quite different,as are the apparent Kds (200 nM and 5 mM for Sa-Lrp [native]and solfataricus Lrs14 [His tagged], respectively) determinedfor binding to their own control region. The oligomeric state ofLrs14 has not been determined, and it is not known whetherthe protein is able to interact with leucine, the major effectorof the regulon in E. coli; the involvement of a potential HTHDNA-binding motif in Lrs 14 and the importance of major andminor groove determinants in complex formation also remainto be investigated. Further experimentation is clearly requiredto allow a more detailed comparison of these two archaealmembers of the Lrp-like family of DNA-binding proteins, todetermine their respective targets, and especially to unravelthe molecular details of their interference with the transcrip-tional apparatus. This latter aspect has been studied in detailfor only one archaeal transcription regulator, MDR1 fromArchaeoglobus fulgidus, that down regulates its own transcrip-tion in a metal-dependent manner and does so by preventingthe recruitment of RNA polymerase and not by abrogating thebinding of TATA-binding protein (2).

FIG. 8. Effect of temperature on binding of Sa-Lrp to the 334-bp promoter-operator fragment. Aliquots (1.6 mg) of purified Sa-Lrp protein were incubatedwith 59-end 32P-labeled DNA for 15 min at 37°C, then shifted to various tem-peratures ranging from 60 to 100°C, and incubated for another 15 min. Sampleswere immediately loaded onto a 5% polyacrylamide gel to separate free DNAmolecules (F) from protein-DNA complexes (B). The position of single-strandedDNA (S) formed by heat-induced strand separation is indicated. The sample inlane 10 was first chilled on ice prior to charging on the gel; this avoids theformation of small amounts of double-stranded DNA by reannealing upon slowcooling, as observed in lane 9.



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ACKNOWLEDGMENTS

We are grateful to J.-P. ten Have, A. Kholti, and C. Tricot for theartwork. We thank P. Falmagne and R. Wattiez at the University ofMons-Hainaut for N-terminal amino acid sequence determinations.

This project was supported by the Fund for Scientific Research-Flanders (FWO Vlaanderen, grants G.0040.96 and G.0069.00) and bya Krediet aan Navorsers (FWO Vlaanderen, grant 1.5.049.99) to D.Charlier.

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