©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Interactions between the Repressor and the Early Operator Region of Bacteriophage Mu (*)

(Received for publication, December 4, 1995; and in revised form, January 21, 1996)

Philippe Rousseau (§) Mireille Bétermier (¶) Michael Chandler Robert Alazard (**)

From the Laboratoire de Microbiologie et Génétique Moleculaires, CNRS, 118 route de Narbonne, 31062 Toulouse Cedex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The repressor of bacteriophage Mu, c, binds to three operator sites, O1, O2, and O3, overlapping two divergent promoters, which regulate the lytic and lysogenic pathways. Its binding to this operator region generates several complexes, which were analyzed by DNase I protection experiments. We demonstrate that c first binds to two 11-base pair partially repeated sequences in O2 that could represent ``core'' binding sites for the repressor. This initial interaction serves as an organizer of a more complex nucleoprotein structure in which O2, O1, and O3 become successively occupied. The quaternary structure of the repressor was also investigated. Size exclusion chromatography and protein-protein cross-linking experiments with chemicals that possess linking arms of various lengths indicate that the repressor oligomerizes in solution. A model is proposed describing the successive interactions of c with the operator sites O2, O1, and O3 leading to the elaboration of a higher order structure in which the early lytic functions are repressed.


INTRODUCTION

The repressor, c, of the temperate bacteriophage Mu controls the choice between lysogeny and lytic development. The decision is exercised at the level of transcription by binding of c to the early operator region located about 1 kilobase from the left end of the phage genome (Fig. 1). This operator is contained within a 200-bp (^1)region composed of two divergent promoters (Pe and Pc) and three operator sequences, O1, O2, and O3. These have been defined by DNase I protection of the region by the repressor (Krause et al., 1983, 1986; Alazard et al., 1992; Goosen et al., 1984). Binding of c to O1 and O2 appears to occur simultaneously at low repressor concentrations and prevents the initiation of transcription at the early promoter Pe. Binding to O3, which requires higher concentrations of c, inhibits transcription of the c gene from the Pc promoter (Goosen et al., 1984; Alazard et al., 1992). A binding site for integration host factor (IHF) is located between O1 and O2. This DNA-binding protein bends DNA and promotes the formation of looped structures (Craig and Nash, 1984; Gamas et al., 1987; Prentki et al., 1987; Stenzel et al., 1987; Robertson et al., 1988; Thompson et al., 1988). IHF plays a complex role in the regulation of the early functions of Mu. It has been demonstrated that it stimulates transcription from Pe, favoring lytic growth (Krause et al., 1986), possibly by direct interaction between bound IHF and RNA polymerase (Goosen and Van de Putte, 1995). On the other hand, IHF stabilizes the interaction among the repressor and O1 and O2 and allows the establishment and the maintenance of lysogeny (Vogel et al., 1991; Alazard et al., 1992; Gama et al., 1992; Bétermier et al., 1995).


Figure 1: Schematic view of the whole genome of Mu and its early operator region. The cross-hatched boxes represent the left (attL) and right (attR) ends of the Mu genome. The unfilled box is the early operon encoding for the proteins: Ner, pA, and pB (lytic cycle). An extended drawing of this region is represented below as unfilled boxes showing the repressor binding sites O1, O2, and O3 and the two early divergent promoters Pe (early lytic genes) and Pc (repressor gene) indicated by arrows pointing in the direction of transcription. The oval symbol represents the specific DNA binding site for the host protein, IHF. The black arrows and the roman numbers locate the imperfectly repeated sequences of 11 bp (5`-CTTTT(NNNA/TA/TA/T)-3`) found several times in each operator site (Krause et al., 1986).



Previous results (Alazard et al., 1992) show that the interaction of c with a linear operator fragment leads to the formation of at least five high molecular weight DNA-protein complexes, which can be separated using gel retardation techniques. The complexes with lower apparent molecular weight can be chased successively into the higher molecular weight forms with increasing c concentrations. DNase I protection experiments of c-operator interactions were not sufficiently precise to determine the nature of each complex. The results indicated, however, that c protects defined areas in the operator region that are larger than would be expected for the binding of a 21-kDa protein monomer (Alazard et al., 1992). An explanation for this is that each operator region is bound by several repressor molecules.

Although no experimental results characterizing the repressor recognition sequence are available, a close examination of the operator region has revealed imperfectly repeated nonsymmetric 11-bp sequences in each of the operator site; O1, O2, and O3 contain 3, 4, and 2 copies, respectively (Krause et al., 1986). These might represent individual binding sites for repressor. Interestingly, Clubb et al.(1994) have shown that a 14-mer oligonucleotide containing a consensus of these sequences is contacted by the DNA binding domain of the phage Mu transposase, a protein that shares significant homology with the repressor in its NH(2)-terminal region and is also able to bind to the operator (Mizuuchi et al., 1989; Leung et al., 1989; Surette et al., 1989).

To obtain a better understanding of the interaction of c with its operator sites, we have determined the DNase I protection patterns of individual gel-purified complexes. Our results indicate that c interacts first with O2 at two of the imperfectly repeated sequences. With increasing repressor concentrations, this initial interaction evolves into a highly organized nucleoprotein structure in which O2, O1, and O3 are successively protected. Our studies also include an analysis of the quarternary structure of c using gel filtration and chemical cross-linking. We present evidence that this protein oligomerizes in solution as a function of its concentration, as described previously for the repressor of the related phage, D108 (Kukolj et al., 1992).

A model described by Vogel et al.(1991) proposes that repressor molecules bound at O1 and O2 interact in a looped structure. The data presented here are fully consistent with this hypothesis. Moreover, they extend the model by suggesting how successive interactions of c with the operator sites O1, O2, and O3 might generate this nucleoprotein structure.


MATERIALS AND METHODS

Bacterial Strains, Plasmids, and Media

Strain MK291 containing plasmid pMK282 (Mizuuchi et al., 1989) was used for the purification of Mu repressor. This plasmid carries the gene for the wild type Mu repressor cloned in the pKC30 vector, under the control of the phage pL promoter (Rao, 1984). pHK09 is a 3.3-kilobase pair plasmid carrying a 289-bp EcoRI-BamHI fragment containing the entire Mu operator region (Higgins et al., 1989).

DNA Manipulations

Large-scale supercoiled plasmid DNA preparation was performed using the alkaline lysis method (Sambrook et al., 1989) or as described (Zerbib et al., 1987). Sequencing reactions were performed according to Maxam and Gilbert (Maxam et al., 1980).

All DNA fragments used as substrates for gel retardation and footprint experiments were amplified from pHK09 by polymerase chain reaction in a Kontron thermal cycler using oligonucleotides listed in Table 1. When necessary, oligonucleotides were end-labeled with [-P]ATP (Amersham Corp.; 5000 Cibulletmmol) using T4 polynucleotide kinase (Biolabs Inc.). Polymerase chain reactions were accomplished by 26 cycles of denaturation (30 s at 95 °C), annealing (2 min at 42 °C), and elongation (1 min at 72 °C). A typical reaction contained 10 pmol of each oligonucleotide, 5 fmol of template plasmid, and 2 units of Biotaq polymerase (Bioprobe Systems) in a total volume of 100 µl. The polymerase chain reaction products were separated by electrophoresis on agarose or polyacrylamide gels and purified, respectively, by cryoelution (Costar Inc.) or as described (Bétermier et al., 1989).



Proteins

Proteins used for calibration in gel filtration and molecular weight markers for electrophoresis on polyacrylamide gels in the presence of SDS (SDS-PAGE) were purchased from Sigma and Biolabs Inc.

Wild type Mu repressor was purified as described (Alazard et al., 1992), and pure Escherichia coli IHF was a generous gift from P. Prentki and colleagues (University of Geneva).

Gel Filtration Chromatography

The apparent molecular weight of native Mu repressor was determined by analytical Ultrogel AcA34 (I.B.F. Sepracor) chromatography. Various amounts of Mu repressor in 100 µl of HED buffer (25 mM HEPES, pH 7.5, 0.4 M KCl, 0.1 mM EDTA, 1 mM dithiothreitol), were loaded onto a 1.6 times 45-cm column, previously calibrated with 1 mg of blue dextran 2000 (2.10^6 Da), beta-amylase (200 kDa), aldolase (158 kDa, 48.1 Å), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa, 35 Å), and ovalbumin (43 kDa, 30.5 Å). Proteins were eluted at a flow rate of 7 ml/h and monitored by UV absorbance at 280 nm. Mu repressor in each fraction was further detected by SDS-PAGE and by gel retardation assays (data not shown). Gel filtration data are presented in terms of K = (V(e) - V(o))/(V(t) - V(o)) versus the logarithm of molecular weights, where V(e) is the elution volume of a given protein, V(o) is the void volume of the column (corresponding to the elution volume of blue dextran), and V(t) is the total volume of the gel bed.

Cross-linking Reactions

Chemical cross-linking employed diepoxybutane (DEB; Aldrich Chemie) and bis(sulfosucinimidyl)suberate (BS^3; Pierce). Assays were performed in a final volume of 10 µl (25 mM, HEPES, pH 7.5, 0.1 mM EDTA, 1 mM dithiothreitol, 0.4 M NaCl). Reactions were initiated by adding DEB (50 mM) or BS^3 (0.1 mM) to 10 µM of repressor. Reactions with DEB were incubated for 30 min or 1 h at 37 °C and stopped by the addition of Tris, pH 7.6, to 250 mM. Reaction mixtures with BS^3 were incubated for 5 and 10 min at 30 °C and stopped by the addition of glycine to 100 mM. 6 µl of each sample was mixed with an equal volume of loading buffer (0.4 M Tris base, 2% SDS, 10% glycerol, 0.04% bromphenol blue, 80 mM dithiothreitol), boiled for 6 min, and analyzed by SDS-PAGE on 8% gels. Protein bands were visualized by silver staining.

Gel Retardation Assays and Footprints of Isolated Nucleoprotein Complexes

Analytical gel retardation assays were performed as described (Alazard et al., 1992), except that divalent cations were omitted. Quantification of the repressor-operator complexes separated on these gels were performed using a FujiX Bas 1000 phosphor-imaging system and TINA PCBAS software. The degree of complex formation was quantified by measuring the amount of complex formed or the residual amount of free DNA.

DNase I footprints of isolated protein-DNA complexes were essentially performed as described (Bétermier et al., 1989). A P-end-labeled DNA fragment (see above) (10^5 cpm; 10-50 ng of DNA) was incubated with 10 or 50 ng of repressor or with 20 ng of IHF in 16 µl of TB buffer, pH 7.5 (25 mM Tris, 70 mM KCl, 1 mM EDTA, 7% glycerol, 50 µg/ml BSA, 1 mM beta-mercaptoethanol). MgCl(2) and CaCl(2) were then added, respectively, to final concentrations of 70 and 30 mM followed by 2 µl of freshly diluted DNase I (final concentration of 0.1 µg/ml). The mixture was incubated at 30 °C for 2 min, and the digestion was stopped by the addition of EDTA (final concentration of 0.05 M). Protein-DNA complexes were separated on a 5% acrylamide gel (Alazard et al., 1992) and visualized by autoradiography. Bands were cut from the gel, and the DNA was eluted by incubation overnight at 42 °C in 500 µl of elution buffer (10 mM Tris, pH 8, 1 mM EDTA, 0.2% SDS, 0.3 M NaCl, 1 µg/ml yeast tRNA). After filtration, the DNA was precipitated by ethanol and resuspended in denaturing loading buffer (80% formamide, 0.01% bromphenol blue, 0.01% xylene cyanol). Approximately 4000 cpm were loaded in each well of an 8% acrylamide, 7 M urea sequencing gel.


RESULTS

Interactions between Repressor and the Entire Mu Operator Region

The five well defined species generated by binding of the Mu repressor to a linear DNA fragment containing the entire operator region are resolved using gel retardation as shown in Fig. 2A (lane 2, bands C1-C5). With increasing c concentration, the least retarded bands are gradually replaced by complexes of higher apparent molecular weight (Alazard et al., 1992). To investigate this complex pattern of interactions in more detail, each of the five species was analyzed by DNase I footprinting (see ``Materials and Methods''). Briefly, after incubation of the repressor with the operator fragment, the binding reaction was treated with DNase I. The different DNA-protein complexes were then separated on a nondenaturing polyacrylamide gel, purified from the gel, and analyzed on a sequencing gel. Typical results are presented in Fig. 2, B and C, and summarized in Fig. 3.


Figure 2: Interactions between the repressor and the complete operator region. A, polyacrylamide gel electrophoresis of operator DNA complexes with repressor or repressor + IHF. A P-end-labeled operator fragment (lane 1) was incubated with 5 ng of repressor (lanes 2 and 3) and with 10 ng of IHF (lane 3). The retarded complexes are identified as C1 to C5 with repressor alone and as I, and IC1 to IC3 when IHF is also included. B and C, DNase I footprinting of the retarded complexes analyzed in A. B and C present results with the lower and upper strands, respectively. Lanes 1 and 2 are the C + T and A + G Maxam and Gilbert sequences of the DNA fragment used. Lane 3, free operator DNA fragment; lane 4, free DNA fragment isolated after incubation with the repressor; lane 5, binding of IHF; lanes 6-10, isolated complexes C1 to C5 shown in A. Operator regions are represented by continuous lines. Positions of increased sensitivity to DNase I are indicated (*). Bases most protected from DNase I cleavage are also indicated (circle). The hypersensitive region between O1 and O2 is represented by the dotted line.




Figure 3: Summary of the footprinting results. The 200-bp operator sequence is shown. Operator sites O1, O2, and O3 and their subsites (I to IX) are represented as in Fig. 1. DNase I protections observed in the different complexes (C1 to C5) are indicated. Shaded area, protection in C1 and C2; open area bordered by dotted line, protection in C3; open area bordered by dashed line, protection in C4 and C5; filled area, hypersensitive region in C2; bullet, site of enhanced cleavage; circle, site of reduced cleavage.



The footprint corresponding to the least retarded complex (C1) formed at the lowest repressor concentrations, shows only a partial protection of O2. Protection is observed on the upper strand at positions 978, 982, 991, and 1010, while enhanced cleavages occur at 986 and 987. Protection extends between positions 999 and 1014 on the lower strand, with amplification at 984 and 993. Thus the first detectable interactions between the repressor and the Mu operator occur in two regions of 14(978-991) and 16 bp (4) in O2 (Fig. 2, B and C, lanes 6). In both cases, only one strand is clearly protected, and the complementary strand is only weakly modified. These protected regions include two 11-bp repeated sequences (IV and VI), which are antiparallel and form part of the quartet of repeated sequences (IV, V, VI, and VII) defining the O2 site (Fig. 3).

The second complex (C2), shows the same protection pattern of O2 but presents strong enhanced cleavages between O1 and O2, from position 938 to 976 on both strands (Fig. 2, B and C, compare lanes 6 and 7), indicative of a significant modification of the structure of this region. A close examination of this footprint also reveals a weak but reproducible modification at two bases (position 912 on the upper strand and 907 on the lower strand) in the O1 region. Thus, this second complex resembles C1 but includes an additional structural modification of the DNA between O1 and O2.

The third complex (C3) shows more extensive protection of O2 and O1 (Fig. 2, B and C, lanes 8). In this species, O2 is protected between positions 978 and 1023 on the upper strand and from 978 to 1028 on the lower strand, while O1 is protected from position 893 to 924 on the upper strand and from 896 to 907 on the lower strand. In addition, the enhancement of DNase I cleavage between O1 and O2 is no longer observed. Thus, from the limited protection of the two 11-bp repeated sequences (IV and VI) of O2 in complex C1, protection is extended outwards in C3 to encompass the remaining two sequences, V and VII. This extensive protection of over 50 bp on O2 and 32 bp on O1 suggests that more than a single repressor molecule may be bound to each of these regions.

In the case of complexes C4 and C5 no difference in the protection pattern was discerned. In both complexes all three operator sequences, O1, O2, and O3 (positions 1054-1071), are protected on both strands (Fig. 2, B and C, lanes 9 and 10). The difference in migration of the two complexes may be due to differences in structure or stoichiometry, which are not accessible to footprint analysis.

Thus, with increasing concentrations of c, O2 is occupied preferentially, followed by O1 and finally by O3. The first interactions between c and O2 take place at sites IV and VI, suggesting that they are high affinity subsites for repressor binding. These interactions induce a structural modification of the DNA between O1 and O2. Although this DNA region is intrinsically bent (Higgins et al., 1989.; Alazard et al., 1992), the binding of c to its operator sites could further enhance such distortion.

Repressor Binding to Different Operator Site Combinations

The above results are clearly consistent with the idea that each of the individual sites, O1, O2, and O3, play specific roles in the elaboration of the overall architecture and function of the Mu early operator. In an attempt to define these roles, we have examined the binding of c to various combinations of the sites.

Binding of repressor to O3 requires more than twice the concentration necessary to protect O1 and O2 (Krause et al., 1986; Alazard et al., 1992), and repression of the early promotor Pe depends only on the binding of c to O1 and O2 (Krause et al., 1986). These observations suggest that O3 may play only a limited role in defining the architecture of the various c-operator complexes. Its role was investigated in a gel retardation assay using c and a linear DNA fragment of 190 bp containing O1 and O2 but deleted for O3. The results presented in Fig. 4A show that the fragment gives rise to several different species of which four are clearly defined (C`1-C`4). These complexes were analyzed by DNase I footprinting, and the results for the upper strand are shown in Fig. 4B.


Figure 4: Interactions between repressor and a partial operator region (O1-O2). A, polyacrylamide gel electrophoresis of the retarded complexes obtained by the binding of repressor to a partial operator region of phage Mu, containing the O1 and O2 regions. A P-end-labeled operator fragment (lane 1) was incubated with 0.1 ng of repressor (lane 2). The retarded complexes are identified as C`1 to C`4. B, DNase I footprinting of the retarded complexes separated in A. Only footprints for the upper strand are shown. Lanes 7 and 8, C + T and A + G Maxam and Gilbert sequences of the DNA fragment used. Lane 1, free DNA fragment; lane 2, free DNA fragment isolated after incubation with repressor; lanes 3-6, isolated complexes C`1 to C`4 described in A. Operator regions are represented by continuous lines. Positions of increased sensitivity to DNase I are indicated (*). Bases most protected from DNase I cleavage are also indicated (circle). The dotted line between O1 and O2 represents the DNA region modified in the C`2 complex.



The footprint of the first complex (C1`) is identical on both strands to that observed with complex C1 (Fig. 2) and indicates partial occupation of O2. The second complex (C2`) also exhibits the same protection profile as its homologue, C2 (Fig. 2) with an identical partial occupation of O2 and a marked increase in DNase I sensitivity between O1 and O2. The third and the fourth complexes (C3` and C4`) exhibit the same protection patterns. They show an extended occupation of O2 on both strands and a limited occupation of O1 (from 890 to 905 and from 911 to 914 on both the upper and lower strands; Fig. 4B and data not shown). This is very similar to that observed with complexes C3, C4, and C5 (Fig. 2). Thus, neither binding of c to O3 nor the absence of the O3 site significantly modifies the pattern of protection of O1 and O2.

The footprinting results presented here raise the possibility that repressor binding to O2 influences binding to O1. This may be reflected in the induced change in the DNA conformation that occurs between these two sites following occupation of O2 but prior to occupation of O1. To investigate this, we have analyzed repressor binding to the isolated operator sites O1 and O2. DNA fragments of 103 and 98 bp carrying the sites O1 and O2, respectively, were generated from the operator region by polymerase chain reaction, end-labeled, and used in gel retardation assays with repressor. Both give rise to three well defined strongly retarded bands (C"1-C"3) and several minor bands (Fig. 5, A and B). The affinity of c for these two sites was quantified using phosphor imaging. The repressor concentration required to retard 50% of each DNA fragment was used as a measure of the relative affinity of the repressor for each operator. The results presented in Fig. 5C show that an approximately 8-fold higher concentration of repressor is required to complex 50% of the O1 fragment than is necessary to complex the same quantity of O2 fragment. Furthermore, DNase I footprinting of the first two retarded complexes (C"1 and C"2) confirmed the preference of repressor for subsites IV and VI in O2 (data not shown).


Figure 5: Binding of the repressor to single operator DNA, O1 or O2. A, polyacrylamide gel electrophoresis separation of the retarded complexes obtained by the binding of repressor to a partial operator DNA of phage Mu, containing the O1 region. 0.25 nM of a P-end-labeled operator fragment (lane 1) was incubated with 0.3 nM (lane 2), 0.6 nM (lane 3), 1.2 nM (lane 4), 2.4 nM (lane 5), 4.8 nM (lane 6), 9.6 nM (lane 7), 19.2 nM (lane 8), 38.4 nM (lane 9), 76.8 nM (lane 10), and 153.6 nM (lane 11) of repressor. B, identical experiment performed with a partial operator DNA fragment containing the O2 region. C, individual sites binding curves determined for the binding of the repressor to the O1 () or O2 (circle) DNA regions. Complexes shown in panels A and B were quantified as described under ``Materials and Methods.''



These results indicate not only that repressor is able to bind each of the operator sites independently but also that O2 has a higher intrinsic affinity for c than does O1.

Mu Repressor Oligomerizes in Solution

The way in which protection of the O1 and O2 operator sites evolves with increasing repressor concentrations must necessitate the binding of several molecules of repressor. An important question is the nature of the repressor molecules intervening in operator binding: is the active repressor molecule a monomer or a multimer? To address this question we have examined the state of Mu repressor in solution. Wild type Mu repressor prepared as described under ``Materials and Methods'' is at least 95% pure. As judged by SDS-PAGE this protein has an apparent molecular mass of 21 kDa, in agreement with the theoretical molecular weight (Goosen et al., 1984). The apparent molecular weight of the native Mu repressor in solution was investigated by gel filtration. At an initial concentration of 30 µM, the protein eluted as a single species with a molecular mass of 130 kDa (Fig. 6, point 1), corresponding to a Stokes radius of 47.5 Å. When the concentration was raised to 100 and 155 µM, the protein continued to elute as a single species but exhibited apparent molecular masses of 200 and 255 kDa, respectively (Fig. 6, points 2 and 3). These molecular masses are consistent with the existence of the protein in an oligomeric or aggregated state.


Figure 6: Apparent molecular weight determination of the repressor. Mu repressor was applied to a ACA34 column at an initial concentration of 30 µM (point 1), 100 µM (point 2) and 150 µM (point 3). A representation of K (calculated as described under ``Materials and Methods'') versus log molecular mass is shown.



To further characterize the ability of the repressor to oligomerize, chemical cross-linking experiments were performed using both DEB and BS^3, which covalently link lysine residues in vitro (Spaeny-Dekking et al., 1995; Staros et al., 1987). The linking carbon chain of these molecules is 4 and 11.4 Å, respectively. Cross-linked proteins were analyzed by SDS-PAGE after heat denaturation (Fig. 7). Although the efficiency of DEB as a cross-linking agent is low, significant amounts of a species migrating with an apparent molecular weight of 2.3 times that of the monomer appear both as a function of time (compare lane 2 with lane 3, and lane 4 with lane 5) and of DEB concentration (compare lanes 2 and 3 with lanes 4 and 5). No forms larger than this dimer could be observed. On the other hand, when BS^3 was used as a cross-linking agent, several high molecular weight species appeared. These migrated with 2.1, 2.3, 3.4, 3.5, 4, 4.3, 5.2, and 6 times the apparent molecular weight of the monomer, suggesting that they represent all multimeric species between dimers and hexamers. Higher order species were not detected under these experimental conditions. The fact that the bands appear as doublets (2.1 and 2.3, 3.4 and 3.5, 4 and 4.4) may reflect different ways of cross-linking the same multimeric species. The fact that their mobilities do not necessarily correlate precisely with the migration expected for molecules containing an entire number of monomers could be explained by the observation that cross-links can change the expected mobility of proteins in SDS-PAGE (Griffith, 1972.; Dong et al., 1995). Moreover, it should be noted that c contains 20 lysine residues, many of which may be capable of reacting to give rise to different combinations of cross-links. We cannot, however, rule out the possibility that these forms reflect different structural dimers of repressor (Chen et al., 1994). Identical results to those obtained with BS^3 were obtained with disulfosuccinimidyl tartrate, a cross-linking agent with a spacer arm length of 6.4 Å (data not shown).


Figure 7: DEB and bis-sulfosuccinimidyl suberate (BS^3) cross-linking of the repressor. Repressor was incubated, respectively, with no (lane 1), 50 mM (lanes 2 and 3), and 100 mM (lanes 4 and 5) DEB and 0.1 mM (lanes 8 and 9) BS^3. Incubation times were 30 min (lanes 1, 2, and 4), 60 min (lanes 3 and 5), 5 min (lane 8), and 10 min (lane 9). Carbonic anhydrase (29 kDa) was used as a monomeric control and incubated, respectively, with 100 mM DEB for 60 min (lane 6) or 0.1 mM BS^3 for 10 min (lane 10). Cross-linking of the subunits of aldolase is also presented as a positive control in lanes 7 and 11 under identical conditions. Standard molecular weight proteins are shown in lane 12. The products were analyzed by SDS-PAGE and stained with silver.



The results of these cross-linking experiments demonstrate that repressor can assume a range of oligomeric forms and that its highest association state detected in these experiments is a hexamer. Results obtained by gel filtration chromatography indicate that at higher repressor concentration, the form obtained depends on the concentration of the protein. Precise definition of the nature of these different forms will require further analysis.


DISCUSSION

The action of Mu repressor, c, at its operator sites appears significantly different from that of ``classical'' phage repressor systems such as those of and 434. The early operator is composed of three nonoverlapping operator sites, O1, O2, and O3, each of which contains a number of 11-bp repeated units thought to represent ``core'' repressor binding sites (Krause et al., 1986). In the model described by Higgins et al.(1989), Pe is repressed when c is bound to O1 and O2. An extension of this model (Vogel et al., 1991) proposed that c binds cooperatively to the three operator sites to form a looped solenoid structure. Such a structure would be maintained by interactions between repressor molecules bound at O2 and O1 and facilitated by the occurrence of a natural bend in the DNA between these two operator sites. This bending is increased and stabilized by binding of IHF to its cognate site in the interoperator region (Alazard et al., 1992.; Gama et al., 1992.; Bétermier et al., 1995).

In the experiments described here, we have investigated in detail the interaction between c and its operator sites by DNase I footprinting of gel-purified c-operator complexes. An interpretation of the nature of the five nucleoprotein complexes, which can be resolved by gel retardation (Alazard et al., 1992) is presented in Fig. 8(A-D). It should be noted that this representation does not imply any particular quarternary repressor structure (i.e., monomeric or multimeric). Repressor binds first to operator site O2 and then to O1 and finally to O3. The absence of O3 does not modify binding of c to O1 and O2. DNase I protection of O2 occurs initially at two subsites (Fig. 8A) covering two of the 11-bp elements (subsites IV and VI with the consensus sequence 5`-TTAC(C/T)(A/G)AAAAGC-3`). These are arranged as inverted repeats and could represent ``core'' binding sites for the repressor. It is not clear whether one or both of these subsites are needed to bind the repressor, but a single copy is not sufficient for stable complex formation. Gel retardation failed to detect any complex between repressor and a 14-bp double-stranded DNA fragment carrying a single 11-bp consensus sequence. (^2)The results highlight the critical role of both O2 subsites IV and VI in initiating formation of the repression complex. It is interesting to note that in a recent study 9 of 13 point mutations selected in vivo for constitutive expression from Pe mapped in O2, and eight of these were located in the two subsites IV and VI. (^3)


Figure 8: A model for the successive occupation of the Mu early operator region by its repressor c. A, B, C and D are representations of the structure of the isolated c-operator complexes C1, C2, C3, and C4. Operator O1, O2, and O3, their subsites, and the promoters are represented as in Fig. 1. Bound repressor molecules are represented as shaded circles. This representation does not reflect any particular quarternary repressor structure. In A we propose that repressor binds alternatively to one of two subsites (IV or VI) of O2. In B, both subsites are occupied and are then capable of interacting with a subsite located on O1 (indicated by the small arrow joining O2 and O1). This is accompanied by bending of the interoperator region, resulting in hypersensitivity to DNase I. In C, all subsites in both O1 and O2 are occupied, representing the repression complex for Pe. This configuration allows stronger interactions between repressor bound at O1 and at O2 (represented by the double-headed arrow). In panel D, additional occupation of O3 is shown.



Subtle changes in the DNase I protection pattern occur during evolution of C1 to C2. While no additional protection of O2 was observed, a slight protection of O1 becomes apparent (positions 907 and 912), and a marked increase in sensitivity to DNase I is visible in the region between the two operator sites. This presumably reflects a change in DNA conformation (Fig. 8B). It should be noted that this region has been demonstrated to assume a curved conformation in the absence of any protein (Higgins et al., 1989.; Van Rijn, A. et al., 1991; Alazard et al., 1992) and that DNA bending is crucial in stabilizing the overall repression complex (Bétermier et al., 1995). The change in DNA conformation in complex C2 may thus be due to a looping process that facilitates the simultaneous binding of Mu repressor to two sites and reflects interactions at a distance between repressor binding subsites present in O2 and O1. The capacity of c to assume an oligomeric state would be expected to favor such concurrent interactions with several distant sites. In this model, complex C1 would reflect a mixed population in which one or the other of the two subsites in O2 is occupied while in C2 both are occupied (Fig. 8, A and B). This double occupation at O2 permits an initial contact with O1, enhancing the curvature and distortion of the interoperator segment and leading to increased sensitivity to DNase I. An alternative explanation, in which the occupancy of O2 alone transmits a conformational change through the interoperator region, seems inherently unlikely since the distance between the border of O2 and the distal end of the affected region (35 bp) is large.

Evolution of the C2 complex to C3 can be viewed as a result of full occupation of both O2 and O1, with the looped structure being maintained by repressor-repressor interactions. Pe would presumably be repressed in this complex (Krause et al., 1986). Such an arrangement would not necessarily involve as strong a distortion of the interoperator segment as in C2 and thus could explain the loss of hypersensitivity to DNase I. Consistent with this idea is the fact that several of the subsites carried by O2 and O1 are separated by an integral number of helical turns, which would facilitate interoperator contacts (Fig. 8C). In particular, subsites I and III in O1 are expected to be located on the same side of the DNA helix as subsites V and VI in O2, whereas subsites II in O1 and IV and VII in O2 should lie on the opposite face. Alternatively, these higher order complexes might represent ``sandwich'' structures in which two DNA-protein complexes are linked side by side. However, competition experiments designed to probe such complexes using mixtures of DNA fragments of different sizes (for a review see Lane et al. (1992)) failed to reveal the existence of such structures (data not shown). The results of gel retardation experiments using DNA fragments containing either operator demonstrated that repressor has an approximately 8-fold higher affinity for O2 than it does for O1. This large difference in affinity could not be detected in footprinting experiments when both sites are present in their natural configuration on the same molecule (Krause et al., 1986; Alazard et al., 1992. This suggests some cooperativity between the two sites, which may be reflected in the transition between complexes C2 and C3 where both O1 and O2 become completely protected without detectable intermediate states. Occupation of O3 would then lead to repression of Pc (Fig. 8D). Confirmation of this model will necessitate determination of the stoichiometry of c in each of the complexes.

The model also explains how IHF might stabilize these complexes (Alazard et al., 1992.; Gama et al., 1992.; Bétermier et al., 1995). In the presence of both IHF and c four complexes can be detected by gel retardation (Fig. 2A, lane 3, and Bétermier et al. (1995)). The first of these complexes (I) shows protection of the IHF binding site, whereas the more retarded species (complexes IC1 to IC3) show, in addition, the successive protection of O2 (IC1), O2 and O1 (IC2), and all three operator sites (IC3) (Bétermier et al., 1995). The order and extent of protection is identical to that observed in the absence of IHF. This suggests that IHF does not modify the relative affinity of c for O1, O2, and O3 but simply stabilizes the interactions between repressor molecules bound at operator sites O1 and O2.

The above model implies that specific protein-protein contacts are involved in the elaboration, and the stability of the repression complex. In initial studies to investigate these potential interactions, we have demonstrated that c is able to oligomerize even in the absence of operator DNA sequence. This is a common feature of many DNA-binding proteins (Steitz, 1990). Although the quaternary structure of the repressor species that binds to the individual operator sites is not yet known, cross-linking experiments reveal several different species. The existence of these multiple forms does not necessarily imply a heterogeneity of repressor multimers in solution. It might reflect an organization of the repressor monomers in the active molecule permitting the formation of several different types of cross-link. With DEB, which has an arm length of 4 Å, low amounts of dimeric forms were detected while with BS^3 or sulfo-DST, which have longer arm lengths (11.4 and 6.4 Å, respectively), additional species appeared. These showed a range of sizes up to that of hexameric molecules. Thus while DEB couples two closely spaced (up to 4 Å) lysine residues presumably at a monomer-monomer interface, BS^3 (or sulfo-DST) react with more distal lysine residues and expand the detection range of oligomeric species. This reveals more extended interactions characteristic of the quaternary structure of the repressor. While we cannot rule out the possibility that the protein does not behave as a globular species, the results of gel filtration studies at repressor concentrations close to 30 µM are consistent with the idea that repressor can assume a hexameric form. However, when the concentration is increased to 155 µM, the apparent molecular weight increases to twice that measured at 30 µM. It is important to note that dynamic light scattering measurements within a concentration range of 125 µM to 800 µM, show not only that repressor solutions are monodisperse (and therefore do not represent aggregates), indicating the presence of a single defined species, but also that the particle size remains constant over this range.^2 A similar effect occurs with the repressor of the Mu-like phage D108, which has also been shown to form high order multimers in a concentration-dependent manner (Kukolj et al., 1992).

The results presented here underline the complexity of the early operator of bacteriophage Mu. They provide an initial characterization of the evolution of repressor binding to the different operator subsites and demonstrate that repressor is capable of undergoing multimerization. Further studies to determine the stoichiometry of repressor binding, to define the minimal repressor recognition sequence, and to analyze the nature of the protein-protein interaction will provide a more detailed understanding of these interactions and the subtle way in which they regulate the early functions of the phage.


FOOTNOTES

*
The work was supported in part by a direct grant from CNRS. Additional support from CNRS (Interface Chimie-Biologie), the ``Ligue Nationale Contre le Cancer'' (Petijean bequest), and the European Union (HM Network Grant 509391) is gratefully acknowledged. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche.

Present address: Laboratoire de Génétique Moléculaire, CNRS, 46 rue d'ULM 75230 Paris Cedex 05, France.

**
To whom correspondence should be addressed. Tel.: 61 33 59 82; Fax: 61 33 58 86; bob{at}ibcg.biotoul.fr.

(^1)
The abbreviations used are: bp, base pair; IHF, integration host factor; PAGE, polyacrylamide gel electrophoresis; DEB, diepoxybutane; BS^3, bis(sulfosucinimidyl)suberate.

(^2)
R. Alazard, unpublished results.

(^3)
L. Desmet and A. Toussaint, personal communication.


ACKNOWLEDGEMENTS

We are grateful to D. Lane, F. Payre, P. Polard, and M. C. Serre for helpful comments on the manuscript and to D. Villa for photography. We also thank L. Desmet, J. Laachouch, and A. Toussaint for communication of results prior to publication and all the members of the transposable elements group for discussions during the course of this work.


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