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J Bacteriol. Jul 2000; 182(13): 3661–3672.

Purification and Characterization of Sa-Lrp, a DNA-Binding Protein from the Extreme Thermoacidophilic Archaeon Sulfolobus acidocaldarius Homologous to the Bacterial Global Transcriptional Regulator Lrp


Archaea, constituting the third primary domain of life, harbor a basal transcription apparatus of the eukaryotic type, whereas curiously, a large fraction of the potential transcription regulation factors appear to be of the bacterial type. To date, little information is available on these predicted regulators and on the intriguing interplay that necessarily has to occur with the transcription machinery. Here, we focus on Sa-lrp of the extremely thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, encoding an archaeal homologue of the Escherichia coli leucine-responsive regulatory protein Lrp, a global transcriptional regulator and genome organizer. 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 leucine independent. 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 be homotetrameric and to bind to its own control region; this binding proved to be leucine independent and was stimulated at high temperatures. Interference binding experiments suggested an important role for minor groove recognition in the Sa-Lrp–DNA complex formation, and mutant analysis indicated the importance for DNA binding of the potential helix-turn-helix motif present at the N terminus of Sa-Lrp. The DNA-binding capacity of purified Sa-Lrp was found to be more resistant to irreversible heat inactivation in the presence of l-leucine, suggesting a potential physiological role of the amino acid as a cofactor.

Compared to the overwhelming amount of information available on mechanisms of basal transcription and its control in Bacteria and Eucarya, relatively little is known about these mechanisms in Archaea, constituting the third primary domain of life (65). The crucial boxA element (now called TATA box) of archaeal promoters strongly resembles the eukaryotic TATA box of polymerase II-dependent promoters (40, 44, 45, 57), the complex multisubunit composition of the archaeal RNA polymerase is reminiscent of those of the eukaryotic homologues, and functional complementation between archaeal and eukaryotic TATA-binding protein and transcription factor TFB (TFIIB in eukaryotes) has been demonstrated (3, 42, 43, 51, 56, 68). Therefore, the major components of archaeal and eukaryotic transcription initiation appear to be fundamentally related. In contrast, archaeal mRNAs most closely resemble their bacterial homologues; they are frequently polycistronic and are relatively unstable, have no introns (except for some tRNA and rRNA genes), bear no 5′ cap site, and have no or only a very short poly(A) tail. Scrutinizing genome sequences has revealed the existence, in archaea and bacteria, of nearly identical proportions of predicted regulatory proteins bearing a potential helix-turn-helix (HTH) DNA-binding motif, reminiscent of bacterial repressors and activators; the predominant class of HTH motifs in archaea is the winged-HTH motif (1). Therefore, Archaea appear to present the intriguing combination of a eukaryotic type of basic transcription apparatus, the activity of which would be controlled by bacterial-type regulatory proteins. This situation must have profound functional and evolutionary implications, and as a consequence, studies on archaeal transcriptional regulation may contribute not only to the deciphering of fundamental mechanisms of transcriptional repression and activation but also to our understanding of microbial evolution. The present state of knowledge calls for the urgent development of model systems for the study of mechanisms of specific and global transcriptional regulation at the molecular level, especially in extreme- and hyperthermophilic archaea. Indeed, at the present time, regulation of archaeal transcription initiation and mRNA stability have been addressed mostly in methanogens and halophiles, representatives of the Euryarchaeota, but very little information is available on extreme- and hyperthermophilic archaea. Moreover, though mobility shift experiments performed 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 transcription initiation sites of specific genes, these potential regulatory elements have not been characterized thoroughly (for a recent review, see reference 33). Only one detailed study has been performed (2) (see Discussion).

Previously, we reported the cloning and identification of Sa-Lrp, a thermophilic archaeal homologue of the eubacterial leucine-responsive regulatory protein Lrp (9), and recently, Napoli et al. (36) reported the identification of Lrs 14 from Sulfolobus solfataricus as an Lrp-like protein that binds to multiple sites in its own control region and therefore might exert autoregulation. However, it is clear that S. solfataricus Lrs14 and Sa-Lrp are not functionally equivalent (see Discussion). In addition, archaeal sequences homologous to Escherichia coli lrp have been reported in Pyrococcus furiosus (16, 32) and in entirely sequenced archaeal genomes (7, 26, 27, 28, 50). However, the corresponding proteins have not been studied. Bacterial members of the Lrp/AsnC family of transcriptional regulators are either specific transcriptional activators or repressors or global regulators that can exert different effects, depending on the target and the presence of a suitable cofactor. Which proteins among the archaeal Lrp-like proteins are specific or global regulators is at present totally unknown. The best-studied member of the bacterial Lrp/AsnC family of prokaryotic transcriptional regulators, E. coli Lrp, is a global regulator that governs the expression of at least 30 genes constituting the leucine/Lrp regulon. The physiological significance is still poorly understood, but from λplacMu insertions, it was estimated that some 50 to 75 targets might exist (8, 37, 38). Lrp frequently superimposes its effects on more local and specific controls; the effect can be negative or positive and in either case requires leucine or is alleviated by it or is leucine independent. The latter prevails in the negative autoregulation of the lrp gene (63). E. coli Lrp is a small, basic, homodimeric protein that frequently binds to several targets in an array. Binding induces a pronounced bending of DNA (62), and Lrp is considered an architectural element that stimulates the formation of specific nucleoprotein complexes and plays a role in the organization of the bacterial genome, in conjunction with other proteins of the histone-like type, as HU, H-NS, and integration host factor. Mutational analysis has indicated that the protein is composed of three functional domains: the N terminus involved in DNA binding, the central domain responsible for transcriptional activation, and the C terminus involved in the response to leucine (41).

Our previous report was on the nucleotide sequence and transcription initiation site of the lrp gene in the extremely acidothermophilic crenarchaeote Sulfolobus acidocaldarius (9). Though the original report stated that the lrp gene (now designated Sa-Lrp) had been cloned from an S. solfataricus DNA bank, further experimentation and amplification of the lrp gene and several other genes from S. acidocaldarius (type strain DSM 639) and S. solfataricus strains P1 and P2 (DSM 1616 and DSM 1617) with oligonucleotides based on the reported sequence and subsequent sequence determination unambiguously demonstrated that the original clone was derived from S. acidocaldarius and not from S. solfataricus. This kind of confusion happened to several groups and arises from incorrect species assignments and the distribution of mixed cultures (67).

Here we demonstrate the monocistronic nature of the Sa-lrp transcript and analyze the effects of growth phase and nutrient availability on its synthesis. We purified and determined the N-terminal amino acid sequence of Sa-Lrp extracted from both the original host and transgenic recombinant E. coli, characterized recombinant protein purified to homogeneity, studied protein-DNA complex formation, and analyzed the effects of temperature and leucine (the major effector of the regulon in E. coli) on protein-DNA complex formation. We also studied the effects of two single-amino-acid substitutions in the potential HTH motif of Sa-Lrp on its interaction with DNA. Therefore, this constitutes one of the first reports on the purification to homogeneity and the characterization to this extent of a potential regulatory protein of thermophilic archaeal origin and its interaction with DNA. The recent development of a purified in vitro transcription assay specific for S. acidocaldarius (4) should facilitate the further functional analysis of Sa-Lrp.


Strains, media, and growth conditions.

S. acidocaldarius (strain DSM 639) was grown 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 · 7H20, 0.25 g of CaCl2 · 2H2O, 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 · 7H2O, 0.25 g of CaCl2 · 2H2O, H2O to 1 liter, adjusted to pH 3.5] supplemented with 0.3% (wt/vol) glucose and 40 μl of a concentrated mineral solution per liter (20). Growth was determined from the apparent absorbance at 660 nm. Growth conditions for E. coli were described previously (19). Genotypes and descriptions of strains and plasmids are given in Table Table1.1. Where indicated, l-leucine (50 μg/ml), kanamycin (35 μg/ml), tetracycline (15 μg/ml), and chloramphenicol (30 μg/ml) were added. Isopropyl-β-d-thiogalactopyranoside (IPTG) was used at 1.0 mM.

Strains and plasmids used in this work

DNA preparations and manipulations.

Plasmid DNA extraction was based on the alkaline sodium dodecyl sulfate (SDS) lysis method (5) and performed with the commercial Nucleobond AX plasmid extraction kits PC20 and PC100 (Promega). Oligonucleotides were purchased from Gibco BRL and EUROGENTEC. Nuclease digestion, ligation, and dephosphorylation and phosphorylation of DNA fragments and oligonucleotides were performed with commercial enzymes and buffers (Boehringer Mannheim) according to the manufacturer's instructions. DNA fragments obtained after endonuclease digestion or by PCR amplification were purified by agarose gel electrophoresis and recovered from the gel by the hot phenol extraction procedure or directly purified on the column (QIAquick PCR purification kit; Westburg). Competent cells were prepared by CaCl2 treatment (15). Enzymatic and chemical DNA sequencing was performed by the methods of Sanger et al. (47) and Maxam and Gilbert (35), respectively.

Oligonucleotide-directed mutagenesis.

The single-amino-acid substitution mutants R44A and L48A were constructed by oligonucleotide-directed mutagenesis using the QuickChange site-directed mutagenesis kit (Stratagene), double-stranded pET24Lsa plasmid DNA as the template, and the complementary pairs of oligonucleotides LrpR44A-LrpR44Arev and LrpL48A-LrpL48Arev (Table (Table2)2) as the primers, according to the manufacturer's instructions. The reaction products were transformed into competent cells of E. coli strain XL1-Blue. Plasmid DNA extracted from individual clones was submitted to enzymatic dideoxy chain-terminating sequencing (47) to verify the presence of the desired mutation and the correctness of the rest of the Sa-lrp gene.

Oligonucleotides used in this work

Reverse transcriptase primer extension.

S. acidocaldarius was grown at 75°C in complex or minimal medium, supplemented with 50 μg of l-leucine per ml when indicated, and arrested either in the exponential phase of growth (A660 of 0.4 and 0.6) or in the stationary phase (A660 of 1.0). Cells were collected from 200-ml cultures by centrifugation, and total RNA was prepared by the Life Technologies procedure using the Trizol reagent (12). Total RNA (25 or 100 μg) was mixed with about 40,000 cpm of 5′-end 32P-labeled oligonucleotide primer (21-mer SalrpRT for Sa-lrp and 22-mer SapyrBRT for pyrB) and after overnight hybridization at 42°C elongated with 10 U of avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim) at 40°C for 1 h, as described previously (9). Chain-terminating DNA sequencing reactions of the noncoding strand obtained with pSPYR3 plasmid DNA as the template and the same 5′-end-labeled oligonucleotides as primer were used as reference ladders.

Reverse transcription-PCR (RT-PCR).

Total RNA was extracted from a 100-ml culture of S. acidocaldarius cells grown in complex medium and harvested in the stationary phase. The RNeasy Midi kit (Qiagen) procedure was used according to the manufacturer's instructions and with inclusion of a supplementary DNase I treatment to remove all traces of possible contamination with DNA. cDNA synthesis was performed with 1.0 μg of RNA, 1.0 mM each of the four deoxynucleoside triphosphates, 1.0 mM dithiothreitol, 30 pmol of oligonucleotide, and 50 U of Expand Reverse Transcriptase (Boehringer Mannheim) in the commercial buffer at 42°C for 90 min in a total volume of 20 μl, and in the presence of 25 U of RNase inhibitor. The reaction was stopped by heating at 95°C for 2 min. cDNA aliquots (3.0 μl) were used as the template in the PCR amplification step with different combinations of oligonucleotide pairs (30 pmol each) in a total volume of 50 μl, with 0.2 mM each of the four deoxynucleoside triphosphates and 1.5 U of PFU DNA polymerase (Promega). Initial denaturation was for 5 min at 94°C, PFU was added at 80°C, and synthesis was performed 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 μl) were analyzed to identify the size and amount of the amplified products by electrophoresis on a 1.5% agarose gel.

Mobility shift electrophoresis.

Mobility shift experiments were performed by the method of Fried and Crothers (17), with modifications. 32P-labeled DNA fragments, labeled at one or both 5′ ends were prepared by PCR amplification with 5′-end 32P-labeled oligonucleotides and purified by polyacrylamide gel electrophoresis. Protein-DNA complexes were formed in 20 μl of Lrp binding buffer (20 mM Tris-HCl [pH 8.0], 0.4 mM EDTA, 0.1 mM dithiothreitol, 50 mM NaCl, 1 mM MgCl2, 12.5% glycerol) with 1 to 5 ng of labeled fragment and in the presence of a 100-fold excess of nonspecific competitor (sonicated herring sperm DNA) for 25 min at 37°C (unless otherwise stated) and loaded onto preelectrophoresed 4 or 5% polyacrylamide gels in TEB buffer (89 mM Tris, 2.5 mM EDTA, 89 mM boric acid). Gels were run in the same TEB buffer at room temperature at 12 V/cm until penetration of the DNA into the gel and then for a further 3 h at 8 V/cm. The binding buffer is similar to the one used for E. coli Lrp (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, did not improve complex formation; lowering the pH even had a slight negative effect. The addition of bovine serum albumin (BSA) (at 50 μg/ml) had no effect. Glycerol, however, had a stabilizing effect (not shown).

DNase I footprinting.

DNase I footprinting experiments with purified recombinant 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 a 2-liter culture of E. coli strain HMS174(DE3)pLysS carrying plasmid pET24Lsa, grown in complex medium supplemented with chloramphenicol and kanamycin and induced with 1.0 mM IPTG at a cell density of 6 × 108/ml for 5 h. Cells were collected by centrifugation, rinsed with extraction buffer (50 mM Tris-HCl [pH 8.0]), resuspended in 15 ml of extraction buffer, and disrupted by sonication in a Raytheon sonicator (250 W) for 25 min at 4°C. Cell debris were removed by centrifugation for 30 min at 30,000 × g. The pellet was discarded, and the supernatant was incubated at 70°C for 5 min, with shaking. An extra 4 ml of extraction buffer was added, and the denatured proteins were removed by centrifugation for 30 min at 30,000 × g. The supernatant was loaded onto a Mono-S HR 10/10 ion exchange column (fast protein liquid chromatography; Pharmacia) preequilibrated with extraction buffer, and eluted with a NaCl gradient (0 to 1.0 M). Fractions containing Sa-Lrp protein (identified by mobility shift electrophoresis) were pooled, concentrated about fourfold on Centricon 10 membrane filters (Amicon), and further purified by gel filtration chromatography on a Superose P12 HR 10/30 column, equilibrated with extraction buffer containing 0.1 M NaCl. Sa-Lrp emerged as a single peak with an apparent molecular mass of 67.0 kDa. Sa-Lrp-containing fractions were pooled, concentrated, and desalted on Centricon filters. The final yield was about 10 mg of Sa-Lrp protein purified to electrophoretic homogeneity. The R45A and L48A mutant Sa-Lrp proteins were purified by the same procedure. As these proteins are severely affected in the DNA-binding capacity, the mobility shift assay could not be used to monitor the protein in the chromotographic steps, and the purification strategy relied entirely on the characteristic behavior of the wild-type protein (see also purification from the native host).

To establish a protocol for purification of Sa-Lrp protein from the native host S. acidocaldarius, we took advantage of the properties of the recombinant protein extracted from E. coli. Cells from a 2-liter culture of S. acidocaldarius strain DSM 639 grown in complex medium and arrested in the stationary phase were harvested by centrifugation at 4°C, resuspended in 15 ml of extraction buffer, and disrupted by sonication in a Raytheon sonicator at 250 W for 30 min at 4°C. Cell debris was removed by centrifugation for 30 min at 45,000 × g. The cell-free supernatant was submitted to an ammonium sulfate precipitation step at 50% saturation and centrifuged for 60 min at 30,000 × 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 × g. The pellet was dissolved in 5 ml of extraction buffer and dialyzed overnight against 1 liter of extraction buffer. The dialyzed protein solution was loaded onto a 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 recombinant protein and SDS-polyacrylamide gel electrophoresis [SDS-PAGE]) were pooled and concentrated ca. eightfold on a Centricon 10 membrane filter and further purified by gel filtration chromatography on a Superose P12 HR 10/30 column equilibrated with extraction buffer containing 0.1 M NaCl. Fractions containing Sa-Lrp (peak near 67.0 kDa and analysis by SDS-PAGE) were pooled and concentrated on a Centricon 10 membrane filter to a volume of 200 μl. When subjected to SDS-PAGE, this material showed two bands. The band corresponding to 16.4 kDa was recovered and subjected to N-terminal amino acid microsequencing and was thus proved to correspond to Sa-Lrp. It was impossible to establish unambiguously the N-terminal amino acid sequence of the major contaminant corresponding to a subunit molecular mass of 10.0 kDa, most likely because the band on the SDS-polyacrylamide gel corresponds to more than one protein.


Modulation of monocistronic lrp mRNA levels in S. acidocaldarius.

Previously we have shown that in S. acidocaldarius cells grown in complex medium, Sa-lrp transcription is driven by a strong and typical archaeal promoter initiated at an A residue located 8 nucleotides (nt) upstream of the ATG initiator codon (9). To determine the effects of nutrients and growth phase on the abundance of Sa-lrp transcription, we performed quantitative primer extension experiments with total RNA extracted from S. acidocaldarius cells grown on complex medium or on minimal medium either devoid of or supplemented with l-leucine and harvested either in the exponential phase of growth, near the end of the exponential phase, or in the stationary phase (see Materials and Methods). The results (Fig. (Fig.1a)1a) indicate that Sa-lrp transcription is about threefold more abundant in the stationary phase than in the exponential phase (compare lanes 1 and 3); this effect is specific, since it was not observed with the same RNA preparations for mRNAs of pyrimidine biosynthetic genes (Fig. (Fig.1b).1b). Transcription of the Sa-lrp gene was repressed approximately twofold in complex medium (Fig. (Fig.1a,1a, compare lanes 1 and 4) but leucine had no detectable effect (lanes 4 and 5). In all instances, Sa-lrp transcription was initiated only at the same, previously identified site (9), indicating that under all conditions examined, transcription was initiated from a single promoter. This was confirmed by RT-PCR experiments which demonstrated, moreover, that the Sa-lrp messenger is monocistronic. Indeed, whereas a strong amplified signal was obtained in the RT-PCR performed with a pair of oligonucleotides (oligonucleotides 1 and 2 [Fig. 1c and d]) corresponding to the 5′ and 3′ ends of the Sa-lrp open reading frame (ORF), no or only a very weak signal was detected when one of the oligonucleotides constituting the pairs was located in orf4 (oligonucleotide 4), preceding the Sa-lrp gene, or downstream of it (oligonucleotide 3), respectively. The extremely weak signal measured with the combination of oligonucleotides 1 and 4 (Fig. (Fig.1d,1d, lanes 3 and 4) indicates the quasi absence (0.6% readthrough) of Sa-lrp mRNA produced by readthrough transcription initiated from pyrimidine gene promoters located upstream (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 cDNA synthesized with oligonucleotide 3 as the primer indicates that the majority of the transcripts do not proceed beyond the Sa-lrp gene and most likely transcription stops at the potential type I transcriptional stop signal (TTTTTATT), located 1 nt downstream of the TAA stop codon (see Discussion also). The densitometric analysis indicated 3.5 and 5.3% readthrough, as measured by amplification of the cDNA with oligonucleotides 2 and 5, respectively.

FIG. 1
(a) Quantitative determination of Sa-lrp transcripts and mapping of the transcription start site by primer extension. Lanes 1 to 3, Sa-lrp primer extension reactions with 25 μg of total RNA extracted from S. acidocaldarius cells grown in complex ...

Overexpression of Sa-lrp in E. coli and purification and characterization of the recombinant protein (Sa-Lrp).

The 155-amino-acid potential coding region of Sa-lrp, including the stop codon, was amplified by PCR using plasmid pSPYR3 bearing a 6.9-kb genomic PstI fragment as a template (9) and ligated into NdeI- and BamHI-digested expression vector pET24a, 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 and corresponding to a 16.2-kDa subunit was detected by SDS-PAGE in cell extracts of IPTG-induced cells bearing this recombinant pET24Lsa plasmid; this value is compatible with the calculated molecular mass of 17,640 daltons deduced from the DNA sequence of Sa-lrp (with omission of the initiator methionine residue [see below]). Recombinant Sa-Lrp was purified to electrophoretic homogeneity (Fig. (Fig.2a)2a) from a 2-liter IPTG-induced culture by a combination of three steps: heat treatment of the cell extract, Mono-S ion exchange chromatography, and gel filtration on a Superose P12 HR 10/30 column (for details, see Materials and Methods). The presence of Sa-Lrp protein in the different fractions was assayed by mobility shift electrophoresis, based on the capacity of Sa-Lrp to bind to its own promoter-operator region (see below). On the molecular sieve column, Sa-Lrp emerged as a peak with an apparent molecular mass of 67.0 kDa (Fig. (Fig.2b).2b). To confirm this value, a mixture of purified Sa-Lrp and BSA (67.0 kDa) was subjected to gel filtration chromatography; the two proteins were unseparable and eluted as a single symmetric peak. In contrast, a mixture of Sa-Lrp and ovalbumin (43.0 kDa) eluted as two separate peaks, Sa-Lrp eluting first (not shown). When subjected to SDS-PAGE, purified recombinant Sa-Lrp migrated as a single 16.2-kDa band (Fig. (Fig.2a,2a, lane 5). Combined, these data are most consistent with the native recombinant protein being a homotetramer. The identity of the purified protein was confirmed by determining the N-terminal amino acid sequence of 13 residues (SDRKKIEIDAIDK), which perfectly matches the amino acid sequence deduced from the DNA sequence of the predicted ORF but lacks the initiator methionine. Methionine removal by E. coli methionyl-aminopeptidase (MAP) depends mainly on the nature of the second amino acid residue in the polypeptide chain; the catalytic efficiency of MAP decreases with increasing length of the side chain (23). The second residue in the Sa-Lrp protein is serine; the efficient maturation of the recombinant protein is therefore in good agreement with the proposed rule, and moreover, serine is one of the most abundant N-terminal amino acids found among cytosolic proteins in E. coli. The same situation prevails for Sa-Lrp protein synthesized in the original host. Sa-Lrp purified from S. acidocaldarius (see Materials and Methods) behaved as a homotetrameric protein of 16.4-kDa subunits, as judged by gel filtration and SDS-PAGE. N-terminal amino acid sequencing of eight residues (SDRKKIEI) unambiguously confirmed the identity of the purified protein, identified its translational start, and indicated absence of the initiator methionine from the mature protein, also in the original archaeal host. Therefore, the purified recombinant protein utilized in further in vitro work has exactly the same amino acid sequence as the protein present in the original host (unless posttranslational modification of Sa-Lrp occurs in S. acidocaldarius).

FIG. 2
(a) Determination of the molecular mass of the recombinant Sa-Lrp subunit and degree of purity of the Sa-Lrp preparation by SDS-PAGE analysis on a 4 to 20% gradient gel. Lanes 1 to 3, 10, 5.0, and 2.5 μl, respectively, of crude extract ...

In vitro DNA binding of Sa-Lrp.

The DNA-binding capacity and specificity of purified recombinant Sa-Lrp were investigated by mobility shift electrophoresis. In the presence of a large excess (>100-fold) of nonspecific competitor (sonicated herring sperm DNA), Sa-Lrp bound to a 334-bp DNA fragment (positions −278 to +56) encompassing the promoter and transcription initiation site of Sa-lrp. Even at the lowest Sa-Lrp concentration (about 140 nM) at which we could detect complex formation under these conditions, Sa-Lrp–DNA complexes hardly penetrated the 4 and 5% polyacrylamide gels (Fig. (Fig.3a).3a). The apparent dissociation constant Kd for Sa-Lrp binding to its own control region, as determined from the half-saturation point in mobility shift experiments conducted 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 the binding assay and in the gel solution and the running buffer, did not significantly affect Lrp binding (Fig. (Fig.3a).3a).

FIG. 3
Detection of Sa-Lrp binding to various potential target sites by mobility shift electrophoresis on 5% polyacrylamide gels to separate free DNA (F) from Sa-Lrp-bound (B) DNA molecules. The concentrations of pure wild-type recombinant Sa-Lrp (in ...

Binding to subfragments of the 334-bp operator fragment was performed with partially RsaI-digested operator DNA labeled at both 5′ ends, resulting in a mixture of three fragments, the intact 334-bp operator fragment and subfragments of 204 bp (−147 to +56) and 130 bp (−278 to −147) (Fig. (Fig.3b).3b). The mobility shift experiment performed with this mixture showed that Sa-Lrp bound to the intact fragment with an approximately fourfold-higher affinity than to the 204-bp subfragment, whereas binding to the 130-bp fragment was nearly undetectable, as judged from the decrease of free DNA bands (Fig. (Fig.3b).3b). Similarly, Sa-Lrp was shown to bind better to the intact 334-bp fragment than to the subfragments of 218 bp (−278 to −61) and 116 bp (−61 to +56) generated by DraI digestion (Fig. (Fig.3c).3c). Sa-Lrp binding to the E. coli and P. furiosus lrp control regions could also be detected, though with an approximately three- and fivefold-lower apparent affinity, respectively (Fig. (Fig.3d3d and e), and binding to the P. furiosus gdh promoter (16) was still weaker (Fig. (Fig.3f).3f). Sa-Lrp appears therefore to bind DNA with but relatively weak sequence specificity.

Effect of a single-amino-acid substitution in the potential HTH motif of Sa-Lrp on DNA binding.

In its N terminus, Sa-Lrp bears a stretch that might fulfill the major requirements of a potential HTH motif (9). Arginine 44 and leucine 48 of Sa-Lrp (starting from serine as position 1 of the mature protein) are highly conserved among bacterial members of the Lrp/AsnC family and related archaeal Lrp-like proteins. By oligonucleotide-directed mutagenesis, we have replaced Arg 44 and Leu 48 of Sa-Lrp with alanine (see Materials and Methods). Mobility shift experiments performed with freshly purified wild-type and mutant proteins and with the 334-bp Sa-lrp control region as the target demonstrated that both substitutions severely impaired DNA binding; no complex formation was detected even at mutant protein concentrations 20-fold higher than the one required to observe 50% binding with wild-type Sa-Lrp (Fig. (Fig.4).4).

FIG. 4
Analysis of wild-type and R44A and L48A substituted Sa-Lrp protein binding to the labeled 334-bp Sa-lrp promoter-operator fragment by mobility shift electrophoresis to separate free DNA (F) from protein-DNA complexes (B). The protein concentrations used ...

Effect of small, groove-specific DNA ligands on Sa-Lrp binding.

Distamycin is a basic oligopeptide of which the three N-methylpyrrole rings interact noncovalently with the narrower minor groove of A+T-rich sequences containing clusters of at least four A−T pairs (14, 58). The addition of increasing concentrations of distamycin in the Sa-Lrp binding assay resulted in a gradual decrease of complex formation, as determined by mobility shift electrophoresis (Fig. (Fig.5a).5a). A significant interference effect was already observed at 5 μM distamycin, and binding was totally abolished at 250 μM. Interestingly, a remarkable effect on migration was also observed for free DNA, indicating extensive binding of distamycin to the Sa-lrp control region and local structural deformation of the double helix. A similar effect of distamycin on the migration of naked DNA was observed with the region upstream of the P1 promoter of the E. coli carAB operon (D. Charlier, unpublished data) and the E. coli pap operon (66). Methyl green binds to hydrophobic surfaces in the major groove. Although this small ligand also affects Sa-Lrp binding, the effect was clearly less pronounced, requiring about 25-fold-higher concentrations of methyl green than of distamycin to obtain a comparable degree of binding interference (Fig. (Fig.5b).5b). Since the DNA-binding affinities of these two ligands have been claimed to be apparently equivalent (29), these results emphasize the importance of minor groove geometry for Sa-Lrp binding. Previously we have reported a similar interference effect of both ligands with binding of the E. coli arginine repressor (61), a member of the winged HTH family of DNA-binding proteins (54) that make contacts to minor and major groove determinants of the operators.

FIG. 5
Interference effect on Sa-Lrp binding by distamycin A and methyl green. End-labeled 334-bp Sa-lrp promoter-operator DNA was incubated at 37°C with 80 μg of Sa-Lrp per ml and increasing concentrations of small, groove-specific ligand. Sa-Lrp-bound ...

DNase I footprinting of Sa-Lrp.

DNase I footprinting of Sa-Lrp protein to a 334-bp DNA fragment bearing the Sa-lrp control region revealed on each strand a very large and not yet well 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 subdivided into several smaller regions of Sa-Lrp-induced protection against nuclease attack separated from each other by short regions (a few nucleotides long) of normal accessibility to DNase I (Fig. (Fig.6).6). Moreover, a few sites hypersensitive to DNase I cleavage were created upon Sa-Lrp binding, whereas the region covering the TATA box was rather resistant to DNase I action, even in the absence of Sa-Lrp. It is well-known that the minor groove of AT-rich sequences is narrow, hence restricting the accessibility to the nuclease. The extent and the complexity of the Sa-Lrp footprint most likely reflect the interaction of more than one protein molecule with the Sa-lrp control region and the formation of a higher-order structure possibly involving DNA bending, looping, and/or wrapping. Further experimentation is required to analyze the details of this complex protein-DNA interaction.

FIG. 6FIG. 6
(a) Part of an autoradiogram of a DNase I footprinting experiment of the 334-bp Sa-lrp promoter-operator region (lower strand labeled) protected with Sa-Lrp. A+G and C+T are the corresponding Maxam-Gilbert sequencing ladders. The DNase ...

Sa-Lrp has an intrinsically thermostable DNA-binding activity.

The functional thermostability of Sa-Lrp was determined by incubating aliquots of purified recombinant protein at 0.6 mg/ml for 15 min at various temperatures from 75 to 100°C in 5°C increments and a subsequent assay of the residual binding capacity to the 334-bp Sa-lrp promoter-operator fragment in a mobility shift electrophoresis experiment. Up to 80°C, no irreversible inactivation could be observed, whereas upon incubation of the protein at 85°C, the residual binding capacity started to decline progressively, and at 100°C, the binding capacity was completely and irreversibly abolished (Fig. (Fig.7a).7a). Interestingly, the addition of l-leucine at 10 mM to the protein solution prior to incubation at a high temperature (92°C) stabilized the protein against heat inactivation (Fig. (Fig.7b).7b). A similar but less pronounced effect could be observed with l-valine, whereas l-alanine had no significant effect. Therefore, the stabilizing effect of leucine appears to be specific and is suggestive of a physiologically relevant interaction of this amino acid with Sa-Lrp protein.

FIG. 7
(a) Effect of heat on the binding capacity of Sa-Lrp. Aliquots of purified Sa-Lrp protein (at 0.6 mg/ml) were incubated for 15 min at various temperatures, chilled on ice, centrifuged for 5 min in an Eppendorf centrifuge, and stored on ice before incubation ...

Sa-Lrp–operator complex formation is stimulated at high temperatures.

To determine the effects of temperature on complex formation and complex stability, identical amounts of purified recombinant Sa-Lrp were incubated in the presence of end-labeled 334-bp operator DNA for 15 min at 37°C, then shifted to various temperatures, incubated for a further 15 min, and immediately loaded on a polyacrylamide gel to separate protein-DNA complexes from free DNA molecules (Fig. (Fig.8).8). The results indicated an increase in complex formation when the temperature was raised from 37 to 60°C, and this effect was even more pronounced at 70 and 80°C. From 90°C on, a decrease could be observed, with complex formation at 90°C still higher than that measured at 37°C. At 95°C, some binding could 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 of the protein and denaturation of the target DNA molecules. The labeled material migrating with a velocity intermediate between those of protein-DNA complexes and free single-stranded DNA observed at very high temperatures (lanes 7 and 8) might represent Sa-Lrp-bound single-stranded DNA molecules or, more likely, partially denatured double-stranded molecules stabilized by bound Sa-Lrp against heat denaturation. Combined, these experiments clearly demonstrate that Sa-Lrp is an intrinsically thermostable DNA-binding protein and that its interaction with the DNA is stimulated at high temperatures and physiological temperatures.

FIG. 8
Effect of temperature on binding of Sa-Lrp to the 334-bp promoter-operator fragment. Aliquots (1.6 μg) of purified Sa-Lrp protein were incubated with 5′-end 32P-labeled DNA for 15 min at 37°C, then shifted to various temperatures ...


In order to understand archaeal molecular physiology and the molecular mechanisms modulating archaeal gene expression, we took advantage of the recently cloned Sa-lrp gene of S. acidocaldarius (9) (also see the introduction), encoding a homologue of the E. coli leucine-responsive regulatory protein Lrp, a global transcriptional regulator and genome organizer, to start investigations on this potential archaeal transcriptional regulator (Sa-Lrp) and its interaction with DNA. Although generally more than one hypothetical regulatory protein belonging to the Lrp/AsnC family of DNA-binding proteins can be recognized in bacterial and archaeal genomes (up to seven in B. subtilis [30]), most of which must be local and specific rather than global regulators, we may have identified a homologue of the global bacterial regulator that might fulfill a similar global function in an archaeon. Without physiological data, which are currently very difficult to gather for thermophilic archaea, this is difficult to prove. Nevertheless, a comparison of Sa-Lrp with the S. solfataricus (type strain P2) genome has revealed the existence of a homologue (c08 044) that shows 74% amino acid sequence identity with Sa-Lrp; moreover, the corresponding genes are located in an identical genomic environment. 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 its homologue from S. solfataricus are the best candidates to fulfill a task similar to that of bacterial Lrp in the Sulfolobales. Moreover, several other lines of evidence are at least compatible with this proposal. (i) There is the relative abundance of Sa-lrp mRNA and protein synthesis, especially for a regulatory protein (difficult to quantify exactly, but in any case the Sa-lrp messenger is at least 10-fold-more abundant than the pyrimidine biosynthetic gene transcripts). (ii) The modulation of Sa-lrp transcription as affected by growth phase and nutrient availability 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 autoregulation. (iv) Most significantly, l-leucine specifically protects the archaeal Sa-Lrp binding capacity against irreversible heat inactivation; this effect likely reflects a physiologically significant interaction of leucine with the Sa-Lrp protein and suggests that leucine might function as the main effector of a hypothetical regulon. In addition, the high predicted (and experimentally confirmed [data not shown]) pI of 8.9 for the S. acidocaldarius protein (9.0 for the S. solfataricus homologue) is characteristic for Lrp proteins, and the subunit length and the absence of tryptophan residues add to the similarity of the bacterial and archaeal Lrp-like proteins. The specific transcriptional regulators of the Lrp/AsnC family appear to be somewhat different. Pseudomonas putida BkdR, a specific transcriptional activator of the bkd operon has a lower pI of 5.89 (34), and the pI of E. coli AsnC, a transcriptional activator of the Lrp-like family is 6.35.

Sa-Lrp is a tetrameric protein, whereas E. coli Lrp is a dimer; this higher oligomeric form of the regulatory protein observed in the extreme thermophilic archaeon might be related to its thermal stability. Indeed, organization into a higher oligomeric form is one of the strategies that nature has developed for stabilizing the native conformation of proteins at high temperatures, as clearly demonstrated for dodecameric ornithine carbamoyltransferase of P. furiosus (60). However, it should be noted that other mesophilic and more distantly related members of the AsnC/Lrp family like Bacillus subtilis LrpC and P. putida BkdR are tetramers as well (55).

In all growth conditions tested (exponential and stationary phase, complex and minimal medium, also supplemented with leucine), Sa-lrp transcription was initiated with an A residue 8 nt upstream of the translational start. Sequence analysis of the upstream region is consistent with the relative abundance of the Sa-lrp messenger. The Sa-lrp promoter bears typical archaeal TATA promoter (TTTAAC) and upstream BRE (transcription factor B recognition element) elements that show a good match to the consensus sequence and are ideally situated with respect to the transcription start site. We have demonstrated that the Sa-lrp transcript is monocistronic and most likely stops at the pyrimidine-rich stretch (TTTTTATT) located 1 nt downstream of the translational stop codon. Transcriptional terminator sequences are not yet well defined in archaea but have been proposed to be of two types in S. solfataricus. Type I terminators (45) consist of a pyrimidine stretch similar to the one proposed here for Sa-lrp. The apparently more frequently occurring type II terminators correspond to the W2TGTATN2W2 consensus sequence (W = 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 overlaps the TATA box (TTTAAC) by 3 nt. This sequence appears to constitute the very efficient terminator signal for one wing of a bipolar pyrimidine operon that precedes Sa-lrp on the genome 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 short Sa-lrp leader preceding the ATG codon, and one could not be recognized downstream of the translational initiation codon. This observation raises the possibility that Sa-lrp messenger is translated in the absence of a conventional ribosome binding site. Apparently, this situation does not constitute a severe handicap for efficient translation; a similar situation has been reported for bacterial, archaeal, and eucaryal genes, even for proteins that are produced in large amounts (25).

We have demonstrated binding of the 67.0-kDa homotetrameric Sa-Lrp protein to its own control region; moreover, this interaction was shown to be leucine independent. Mobility shift electrophoresis, DNase I footprinting, and small ligand binding interference experiments indicated that a large DNA region overlapping the promoter elements is involved in Sa-Lrp–DNA interaction and that the minor groove of the DNA helix is particularly important for complex formation. Negative autoregulation of E. coli lrp is also independent of leucine and involves the binding of several protein molecules over a distance covering more than 200 bp.

The stimulation of Sa-Lrp–DNA complex formation observed at high temperatures, up to 80°C at least, emphasizes the thermophilic character of this interaction. In conjunction with the intrinsic thermostability of the protein, its increased functional thermotolerance observed in the presence of leucine (suggestive of a specific interaction of Sa-Lrp with this amino acid [the major effector of the E. coli Lrp regulon]), and the abundance, these observations suggest that Sa-Lrp may fulfill the role of a global transcriptional regulator of a hypothetic regulon in this extremely thermophilic crenarchaeote.

E. coli Lrp bears in its N-terminal part a potential HTH motif (66) that from a mutational analysis has been proposed to be responsible for binding to DNA (41). The equivalent region in the Sulfolobus protein might very well adopt a similar fold, as most of the major requirements of a HTH motif (6) are fulfilled (discussed reference 9). The severe reduction in DNA-binding capacity observed in the single-amino-acid substitutions R44A and L48A located in this region lends further support to the existence of this motif and its importance for DNA binding. There is as yet no structural model available for any of the bacterial or archaeal members of the Lrp-like family of DNA-binding regulatory proteins. A more detailed interpretation of the observed effects is therefore premature, and unfortunately, the lack of well-developed molecular tools for thermophilic archaea strongly limits the functional analysis in vivo. However, the present evidence gathered on Sa-Lrp is sufficient to warrant further studies on this DNA-binding protein. Attempts to grow crystals for the structure determination by X-ray diffraction are in progress (in collaboration with D. Maes, Ultrastructure Department, Vrije Universiteit Brussel, Brussels, Belgium), and in vitro transcription assays will be performed to gather further evidence for a physiologically relevant regulatory function for the archaeal regulator (in collaboration with S. Bell and S. Jackson, Cambridge University, Cambridge, United Kingdom).

Recently, Napoli et al. (36) presented the Lrs 14 protein of S. solfataricus as an Lrp-like protein that binds to its own control region. Lrs 14 and Sa-Lrp share only 10.3% amino acid sequence identity; this rather weak similarity and sequence comparisons bringing to light the existence of a real homologue (see above) make it very unlikely that Lrs 14 and Sa-Lrp would be functional equivalents. Though both proteins show a growth stage- and nutrient composition-dependent synthesis that is reminiscent of that of E. coli Lrp, the pIs of the two proteins, 8.9 and 8.2 for the S. acidocaldarius and solfataricus proteins, respectively (9.1 for E. coli Lrp), are quite different, as are the apparent Kds (200 nM and 5 μM for Sa-Lrp [native] and solfataricus Lrs14 [His tagged], respectively) determined for binding to their own control region. The oligomeric state of Lrs14 has not been determined, and it is not known whether the protein is able to interact with leucine, the major effector of the regulon in E. coli; the involvement of a potential HTH DNA-binding motif in Lrs 14 and the importance of major and minor groove determinants in complex formation also remain to be investigated. Further experimentation is clearly required to allow a more detailed comparison of these two archaeal members of the Lrp-like family of DNA-binding proteins, to determine their respective targets, and especially to unravel the molecular details of their interference with the transcriptional apparatus. This latter aspect has been studied in detail for only one archaeal transcription regulator, MDR1 from Archaeoglobus fulgidus, that down regulates its own transcription in a metal-dependent manner and does so by preventing the recruitment of RNA polymerase and not by abrogating the binding of TATA-binding protein (2).


We are grateful to J.-P. ten Have, A. Kholti, and C. Tricot for the artwork. We thank P. Falmagne and R. Wattiez at the University of Mons-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 by a Krediet aan Navorsers (FWO Vlaanderen, grant to D. Charlier.


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