©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Participation of the Flank Regions of the Integration Host Factor Protein in the Specificity and Stability of DNA Binding (*)

(Received for publication, April 17, 1995; and in revised form, May 30, 1995)

Laurence Zulianello Peter van Ulsen Pieter van de Putte Nora Goosen (§)

From the Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2300 RA Leiden, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The heterodimeric integration host factor (IHF) protein is a site-specific DNA-binding protein from Escherichia coli that strongly bends the DNA. It has been proposed (Yang, C., and Nash, H. A.(1989) Cell 57, 869-880; Granston, A. E., and Nash, H. [Medline] A.(1993) J. Mol. Biol 234, 45-59; Lee, E. C., Hales, L. [Medline] M., Gumport, R. I., and Gardner, J. F.(1992) EMBO J. 11, 305-313) that the wrapping of the DNA around the protein is stabilized through interactions between the flanks of the protein and the DNA. In order to elucidate which domains of the IHF protein are involved in these interactions, we have constructed mutant proteins in which the C-terminal part of one of the subunits has been deleted. We observed that the C-terminal 3 helix of HimD is involved in the stability of DNA binding, but not in the specificity. In contrast the corresponding 3 helix of HimA is essential for the sequence specificity, since an IHF mutant lacking this domain only binds to the DNA in a nonspecific way. The possible role of the two C-terminal -helical structures in complex formation will be discussed.

We also examined the properties of an IHF mutant that has an amino acid substitution between sheets 1 and 2 of the HimD subunit (R46H). The occupancy of the ihf site by the mutant and wild type proteins differ in the 3` part of the ihf site and as a result the bend introduced in the DNA by the mutant protein is less pronounced. We propose that the arginine 46 in the HimD subunit is in vicinity of the TTR region of the consensus and that through contacts within the minor groove the DNA bend introduced by IHF is stabilized.


INTRODUCTION

Integration host factor (IHF)()is a sequence-specific DNA-binding protein, first identified as an accessory protein for the integration of DNA in the chromosome of Escherichia coli(4, 5) . The protein plays also an important role in a variety of cellular processes, such as transposition, DNA replication, DNA packaging, and control of gene expression (for reviews, see (6, 7, 8) ). IHF is a heterodimeric protein composed of two non-identical but homologous subunits, ((11) ; 35 kDa) and ((10) ; 65 kDa), encoded by the himA and himD genes(9, 10) . These subunits are small basic proteins that also share homologies with the subunits of the E. coli HU protein, another member of the group of histone-like proteins to which IHF belongs(11) . Although IHF can replace HU in some processes (12, 13) , the binding properties of the two proteins are different. HU binds the DNA in a nonspecific way, and multiple protomers can bind to a DNA fragment with about one dimer for each 9 bp(14) . In contrast, IHF recognizes a specific sequence: WATCAANNNNTTR()(15, 16) , and only one IHF protein is bound per ihf site protecting a DNA sequence of approximately 30 bp(1) .

Both HU and IHF are DNA-bending proteins. The crystal structure of the HU protein of Bacillus stearothermophilus has been resolved (17, 18) . From this three-dimensional structure, a model has been proposed for interaction of HU with the DNA. In this model, the two exposed -ribbon arms encircle the DNA. Then through protein-protein interactions between the adjacent domains of the bound protomers of HU, a bend is introduced in the DNA. Combining the structural model for HU and the footprint data of IHF, the following model for the binding of IHF to DNA has been proposed(1) ; the extended arms bind to the DNA through interactions within the minor groove, and in addition, the DNA flanking these minor groove regions interact with the body of the protein to stabilize the pronounced bend which has been estimated to be over 140°(19) . In this way, the IHF protein makes contact with the DNA beyond the consensus sequence and these surrounding sequences have been shown to be important for the stability of the IHF-DNA complex (16, 20, 21, 22) . Active ihf sites are often found in A/T-rich sequences, which could help the bending of the DNA.

From the binding studies with homodimers of HimA and HimD (23, 24) and with mutant IHF molecules(2, 25) ()and from the analysis of suppressor mutants of IHF that recognize mutated ihf sites(3) , it became clear that the arm of the HimA subunit is in direct contact with the WATCAA part of the consensus while the flank of the HimD subunit interacts with the TTR sequence of the consensus(3) .

In this paper, we analyzed which parts of the HimD protein might be responsible for a direct contact with the consensus. We present evidence that the turn between sheets 1 and 2 interacts with the TTR part of the consensus and that this interaction is important for the bending of the DNA.


EXPERIMENTAL PROCEDURES

Strains and Media

Strains AB1157, PP1953 (AB1157, himD), and PP1954 (AB1157, himA) have been described previously(26) .

Bacterial strains used in this study for overproduction of proteins were derivatives of E. coli B strain BL21 in which the T7 RNA polymerase gene is under the control of the lac promoter(27) . In this strain, the himA and himD mutations were introduced by means of P1 transduction using PP1953 and PP1954 as donor strains, resulting in PP2895 (ompT, rM, himA::Tet) and PP2896 (ompT, rM, himD157, Tet insertion next to himD gene). The himA himD double mutant was constructed by P1 transduction using K2704 (10) as donor and PP2895 as an acceptor resulting in PP3363 (ompT, rM, himA::Tet, himD::Cam). All strains were grown in Luria broth (28) containing the appropriate antibiotics (80 µg/ml ampicillin and 25 µg/ml chloramphenicol).

Plasmid Constructions

The HimA- and HimD-overproducing plasmids pGP1138, pGP1104, and pGP1149 have been described previously (23) .

The HimA8- and HimD4-overproducing plasmids were constructed by the introduction of a stop codon at position 273 and 270 in the himA and himD reading frames, respectively. The oligonucleotides CTGTAGACGGATCCTTAGACCCGGCTTTTTAAC and CGGGAGACGGATCCTTAGGCGCGATCGCGCGAGT were used for this site-directed mutagenesis by polymerase chain reaction. The amplified products were cloned under the control of the T7 promoter in pGP1104 and pGP1149 resulting in pGP1311 and pGP1312, which produce proteins without 7 or 4 amino acids of the C terminus of HimA and HimD, respectively. In a similar way, the HimA15- and HimD10-overproducing plasmids were obtained after polymerase chain reaction mutagenesis using primers AACCCGGCTTGTCGACTACTGCCCGGGTC and CTGTAGACGGATCCTTATTTACCAGGTTTAAAG to introduce a stop codon at position 255 of the coding sequence of himA and at position 252 for himD, resulting in pGP1309 and pGP1310, which express the genes under the pT7 control. The subunits that are encoded lack 15 and 10 amino acids of the C terminus of HimA and HimD, respectively.

The HimD-R46H mutant (Fig. 1) was obtained from a collection of randomly isolated IHF mutants.()For the construction of the R46H-overproducing plasmid, the NdeI-BamHI fragment containing himD with the corresponding mutation was inserted in pGP1149(23) .


Figure 1: C-terminal parts of different HU and IHF subunits. A, alignment of the 3 helices and C-terminal extensions of HimA and HimD proteins from E. coli with homologous regions in the HU and IHF subunits from different organisms. B, HimA and HimD mutants used in this study. Only the C-terminal half of the proteins with the predicted 3 helix, sheets 1, 2, and 3, and the arm region are shown. The arrows indicate the end parts of the C-terminal deletions used in this study. Also indicated is the mutation R46H, in the turn between sheets 1 and 2 in the HimD subunit.



Plasmid pGP133, containing the ihf site of bacteriophage Mu, which was described previously by van Rijn et al.(26) , was used for retardation assays and footprint experiments.

Protein Purification

After overproduction, HimD and its derivatives form aggregates in the cells which can be dissolved in urea. Therefore, all dimeric proteins were purified by mixing the lysates of strains in which the separate subunits were overexpressed in buffer B (50 mM Tris, pH 7.5, 1 mM EDTA, 10% glycerol, containing 100 mM KCl and 8 M urea). The lysates were subsequently dialyzed in buffer B containing 100 mM KCl to remove the urea and to allow the reconstitution of the heterodimer.

The HimA8-HimD dimer was purified by mixing the lysates from PP2895 with pGP1311 and with pPG1149; the HimA-HimD4 heterodimeric protein was purified by mixing the lysates from PP2896 with pGP1138 and with pGP1312; in the same way, the HimA15-HimD protein was purified from mixed lysates from PP2895 containing the plasmids pGP1309 and pGP1149, while HimA-HimD10 was isolated from PP2896 with the plasmids pGP1138 and pGP1310. The HimA-HimDR46H (R46H) was purified from PP2896 containing pGP1140 and the HimDR46H-overproducing plasmid.

The purification of the proteins was as described for wild type IHF (23) using P-11 phosphocellulose, heparin, and FPLC-MonoS columns. During purification, the HimA8-HimD and HimA-HimD4 dimers behaved like IHF while the other mutants were eluted at lower salt concentrations.

Gel Retardation Assay

Plasmid pGP133 was digested with HindIII (Pharmacia Biotech Inc.), providing a 180-bp DNA fragment of the regulatory region of phage Mu with the ihf binding site (Fig. 2). The DNA was end-labeled using the Klenow fragment of DNA polymerase (Pharmacia) and [-P]dCTP (3000 Ci/mmol; Amersham Corp.). As a control fragment the 200-bp EcoRI-HindIII fragment of Mu, which is adjacent to the ihf-containing fragment, was used (Fig. 2). To avoid contamination with the 180-bp fragment, pGP133 was first digested with HindIII and the vector fragment was separated from the 180-bp fragment. Next the isolated vector fragment was digested with EcoRI. The deletion of the TTR sequence of the consensus was obtained by digesting the 180-bp HindIII fragment with MluI, followed by filling in of the sticky ends by Klenow enzyme (Pharmacia).


Figure 2: DNA fragments used in this study. A, the 180-bp HindIII fragment of the regulatory region of the phage Mu genome with the ihf binding site and the flanking 200-bp EcoRI-HindIII fragment which is used as the control. Part of the sequence containing the ihf site is indicated. B, the 140-bp fragment containing a truncated ihf site obtained after restriction with MluI. C, the 140-bp fragment containing a truncated ihf site obtained after restriction with MluI and subsequent treatment with Klenow polymerase. The relevant sites are; E, EcoRI; H, HindIII; C, ClaI; M, MluI. The consensus sequence of the ihf site is underlined. The positions with respect to the left end of the Mu sequence are indicated.



Dilutions of the proteins were made in buffer B containing 100 mM KCl. Binding reactions (10 µl) consisted of 3-5 fmol of DNA and the indicated amounts of protein in 50 mM Tris, pH 8.0, 75 mM KCl, 1 µg of bovine serum albumin, and 1 mM dithiothreitol. The reaction mixtures were incubated for 10 min at room temperature, after which 2 µl of loading buffer (25% Ficoll containing 0.04% of bromphenol blue) was added, and subsequently the samples were loaded on to a 5% polyacrylamide gel, which was run at 4 °C. The gels were dried, and the DNA was visualized by autoradiography.

DNase I Footprint Experiments

ClaI-HindIII fragments corresponding to the Mu sequence from position 818 to 1004 (Fig. 2) were labeled at the 3`-end of the HindIII site using the Klenow fragment of DNA polymerase I and [-P]dCTP. The labeled fragments were purified on a 5% polyacrylamide gel. Reactions of 20 µl containing the labeled fragment and different concentrations of protein were incubated for 10 min. at room temperature in 50 mM Tris, pH 7.5, 75 mM KCl, 10 mM MgCl, 3 mM CaCl, 1 mM dithiothreitol, and 100 µg/ml bovine serum albumin. Then 0.01 unit of DNase I (Pharmacia) was added and the reaction mixture was incubated for an additional 5 min before it was quenched with 11 µl of the stop solution (1 µg/µl calf thymus DNA, 200 mM sodium acetate, pH 8.0, 75 mM EDTA). Dried samples were resuspended in sequence loading buffer (80% formamide, 0.1% bromphenol blue, 0.1% xylene cyanol) and electrophoresed on 6% polyacrylamide sequence gel. The gels were dried, and the footprint patterns were visualized by autoradiography.


RESULTS

IHF That Lacks the 8 C-terminal Amino Acids of HimA or 4 C-terminal Amino Acids of HimD Shows Normal DNA Binding

When the amino acid sequences of the HimA and HimD subunits are compared to those of the subunits of the homologous HU protein, it appears that the C-terminal parts of HimA and HimD are more extended (Fig. 1). Whereas the HU subunits contain 1-3 amino acids beyond the C-terminal helix, the HimA and HimD proteins from E. coli have 8 and 4 residues in this region, respectively. The subunits of IHF from Serratia marcescens and Rhodobacter capsulatus also possess such extended C-terminal regions. To investigate whether the extended C-terminal regions participate in the binding specificity of IHF, we have deleted these regions (Fig. 1) by introduction of a stop codon in the reading frames of the himA and himD genes. The proteins have been purified as heterodimers: HimA8-HimD and HimA-HimD4 (Fig. 3), according to the procedure described under ``Experimental Procedures.'' Throughout purification, both truncated proteins behaved as the wild type protein and exhibited the same elution pattern as IHF.


Figure 3: Tricine protein gel of the purified proteins. 1, HimA-HimD4; 2, HimA-HimD10; 3, wild type IHF; 4, R46H; 5, HimA8-HimD; 6, HimA15-HimD.



We have examined the DNA binding properties of the truncated IHF proteins by gel retardation experiments using a 180-bp HindIII fragment that contains the ihf sequence of the regulatory region of bacteriophage Mu. Fig. 4shows that HimA8-HimD and HimA-HimD4 can still stably bind to the DNA. The affinity for the DNA might be slightly reduced for the mutants (a factor of 4) but they still bind to the ihf site in a very specific way. Moreover, the mutant protein-DNA complexes migrate at the same position within the gel as the wild type IHF-DNA complex, indicating that the bends introduced in the DNA by the truncated proteins or IHF are similar. The doubly truncated protein (HimA8-HimD4), that lacks both C-terminal extensions, also stably binds the DNA with a somewhat reduced affinity (data not shown). These results point out that the C-terminal extensions of HimA and HimD do not seem to be essential for the recognition of the ihf site or for the bending of the DNA.


Figure 4: Autoradiograph of retarded protein-DNA complexes with HimA8-HimD (A) and HimA-HimD4 (B). The lower band corresponds to the free 180-bp DNA fragment containing the ihf site of phage Mu. The middle band corresponds to the IHF-DNA complex and the band in the upper part of the gel corresponds to the 4-kb vector fragment of the plasmid used. The amount of proteins are indicated in pmol.



The Deletion of 15 C-terminal Amino Acids of HimA Results in a Loss of Specificity of Binding

Next, we explored the contribution of the C-terminal 3 helices of HimA and HimD to the DNA binding since the position of the corresponding helices in the HU protein suggests that they might be candidates for interacting with the DNA, possibly thereby stabilizing the bend of the DNA (Fig. 9). First we constructed a himA deletion mutant that encodes a protein lacking the C-terminal 15 amino acids comprising the complete last helix (Fig. 1), and we purified the HimA15-HimD heterodimer (Fig. 3).


Figure 9: Schematic representation of the IHF protein and the phage Mu ihf site. The consensus sequence of the ihf site is in bold, and the DNA corresponding to the positions of the H` site that could be cross-linked to the flank of HimA and the arm of HimD (33) are underlined in the upper and lower strand, respectively. The subunits of IHF are indicated separately, with the HimD subunit in bold. Also indicated is the mutation R46H, in the turn between sheets 1 and 2 of HimD.



The binding capacity of the mutant IHF protein that lacks the last helix of the HimA subunit was analyzed by a gel retardation assay with the 180-bp HindIII fragment. As shown in Fig. 5, no specific complex with the DNA could be detected using the mutant protein. Only at very high protein concentrations, a low mobility complex was observed, probably representing the nonspecific binding of the mutant protein to the DNA since at the same concentrations also the vector fragment is shifted in the gel. To test this, we analyzed the binding of the wild type and mutant IHF to a 200-bp EcoRI-HindIII fragment of phage Mu DNA (Fig. 2), adjacent to the ihf-containing fragment and which does not contain an IHF binding site. As seen in Fig. 5B, IHF can bind to this DNA fragment, but a much higher concentration of protein is needed to observe this nonspecific binding as compared to the specific binding. Moreover, the binding affinities of the wild type and mutant protein for the nonspecific DNA are the same.


Figure 5: Autoradiograph of retarded protein-DNA complexes with the HimA15-HimD mutant. Proteins were incubated with the 180-bp HindIII fragment containing the ihf site (A) or with the 200-bp fragment without the ihf site (B). The DNA fragments were prepared as described under ``Experimental Procedures.'' The amounts of protein are indicated in pmol.



We conclude from these results that although the mutant protein exhibits no preferential binding to the ihf target sequence, its general DNA binding capacity is not lost. This may indicate that, in the absence of the C-terminal 15 amino acids of the HimA subunit, the overall conformation of the HimA15-HimD protein still resembles that of the wild type IHF.

Deletion of 10 C-terminal Amino Acids of HimD Affects the Affinity but Not the Specificity of DNA Binding

To investigate the role of the last helix in HimD, we constructed a himD deletion mutant encoding a truncated protein that lacks the C-terminal 10 amino acids, including the 3 helix. The HimA-HimD10 heterodimeric protein was purified (Fig. 3), and the binding to the ihf-containing fragment was examined (Fig. 6). In contrast to IHF lacking the 3 helix of HimA, IHF lacking the 3 of HimD does show specific binding to the ihf site, although the affinity is reduced with respect to wild type IHF by about a factor of 50-100. The DNA-mutant complex migrates at the same position as the wild type complex, indicating that the DNA bend is also not affected by the absence of the 3 helix of HimD. In addition, from footprint analysis (data not shown) it appears that the occupancy of the ihf region by the mutant and wild type proteins are identical again suggesting that they form similar complexes. Apparently, the C-terminal part of HimD is not essential for recognition of the ihf site and for formation of the proper complex. The reduced binding affinity of the mutant protein, however, indicates that the last helix of HimD does play a role in the stability of DNA binding. This might be accomplished through direct interaction with the DNA, or through stabilization of an other DNA binding domain of the IHF protein.


Figure 6: Autoradiograph of retarded protein-DNA complexes with the HimA-HimD10 mutant. Proteins were incubated with the 180-bp HindIII fragment containing the ihf site. The amounts of protein are indicated in pmol.



Mutation R46H in HimD Affects the Interaction with the TTR Part of the Consensus

On the basis of the model of the HU structure, an other prominent candidate for interaction with the DNA can be indicated: the turn between sheets 1 and 2 of both subunits. From our collection of randomly isolated IHF mutants,() one mutant protein, which has a substitution in this region of the HimD subunit (R46H), was chosen for further analysis. The protein was purified as a dimeric protein (Fig. 3), and the binding property of the mutant was analyzed with a retardation assay using the 180-bp HindIII fragment. At the salt conditions that are normally used in our DNA binding experiments (75 mM KCl), the mutant protein binds with an extremely low efficiency to the ihf site (Fig. 7A). To observe a comparable binding as the wild type IHF, about 400 times more mutant protein is needed. The affinity is increased at low salt conditions (0-10 mM KCl), but it is still about 10 times lower than that of wild type IHF (Fig. 7B). Surprisingly, however, we observed in both cases that the R46H-DNA complex migrates faster in the gel than the wild type IHF-DNA complex. Since it has been shown that the migration of the protein-DNA complex in a gel is correlated to the degree of bending introduced in the DNA(29) , this could indicate that the DNA in the mutant complex is bent to a lesser extent. To analyze the mutant protein-DNA complex further, we performed a DNase I footprint with R46H on the upper strand of the 180-bp fragment that contains the ihf sequence (Fig. 8). In the presence of R46H, a clear protection of the DNA is observed but only in the absence of KCl and at higher concentration of protein as compared to the wild type. No protection of the ihf-containing fragment was detected in the presence of 75 mM of salt (data not shown). The binding of the mutant in absence of salt might indicate a defect in electrostatic interactions, which could stabilize either the protein conformation or the DNA-protein complex. As shown in Fig. 8, the protection patterns of the mutant and wild type proteins are very similar, except that one nucleotide, at the 3` side, is no longer protected by the mutant protein. This unprotected nucleotide maps at position 962 of the DNA sequence (Fig. 2). This position is close to the TTR region of the ihf consensus, which might indicate that the R46H mutation affects the interaction of the IHF protein with this region, and as result the DNA could be bent to a lesser extent.


Figure 7: Autoradiograph of the retarded protein-DNA complexes with the R46H mutant. Proteins were incubated with the 180-bp HindIII fragment containing the ihf site in presence of 75 mM KCl (A), the 180-bp HindIII fragment in absence of salt (B). Wild type IHF (C) and R46H (D) were incubated with the truncated 140-bp fragment that lacks the TTA sequence of the ihf site. The amounts of protein are indicated in pmol.




Figure 8: DNase I footprint of IHF and R46H. The positions with respect to the left end of the Mu sequence are indicated. The arrow indicates the cleavage site that is no longer protected by the R46H mutant protein.



To investigate whether indeed the -turn of HimD interacts with the 3` part of the ihf consensus sequence, the TTA sequence was deleted from the Mu ihf site. This was achieved by cutting of the 180-bp DNA fragment with MluI, leading to a fragment of 140 bp (Fig. 2). When the DNA fragment was used without a subsequent Klenow treatment, no binding of the wild type protein could be obtained (data not shown), whereas, as shown in Fig. 7C, after filling in of the recessive ends, IHF binds specifically to the truncated ihf sequence but with a lower affinity as compared to the intact sequence. This result suggests that the CGCG end of the fragment has to be double stranded in order to form a specific complex. The complex with the truncated ihf site is less retarded as compared to the complex with the 180-bp fragment. It is difficult to show whether the bending of the DNA is changed, since the ihf site is now located at the end of the fragment, where the bending contributes much less to the mobility in the gel(20, 21) .

Mutant R46H is also capable to bind to the truncated ihf site that was obtained after Klenow treatment (Fig. 7D) but again in the absence of KCl. Under these conditions, the wild type and mutant proteins bind with comparable affinities to the DNA fragment lacking the TTA part of the consensus. This could indicate that the R46H mutant is specifically disturbed in interaction with this DNA region.

In conclusion, our results point out that the turn between sheets 1 and 2 of HimD might be in close contact with the TTR part of the consensus and, as judged from the retardation assay, this interaction might contribute to the bending of the DNA.


DISCUSSION

According to the recent model that is based on both genetic and biochemical analyses(1, 2, 3) , IHF binds to the DNA through minor groove interactions between the arm of HimA and the WATCAA part of the consensus and the arm of HimD and the 5` side of the consensus. The body of the IHF protein makes additional contacts with the DNA flanking the arm-binding region, and these contacts probably stabilize the bend that is introduced in the DNA. In this paper we present evidence that the turn between sheets 1 and 2 of HimD interacts with the 3` part (TTR) of the consensus. The DNase I footprint pattern of a mutant protein, which contains a substitution in this turn-region of the HimD subunit (R46H), shows that the interaction of this IHF mutant with the 3` part of the consensus is partly disturbed. As a consequence the DNA seems to be bent to a lesser extent by the mutant as compared to the wild type, as judged from the faster mobility of the mutant complex in a gel retardation assay. Although the R46H substitution also seemed to affect the conformation of the protein when incubated at high salt conditions, its direct effect on the interaction with the 3` part of the consensus became clear when gel retardation was performed with the Mu ihf site in which the 3` TTA sequence was deleted. The wild type and mutant IHF proteins bind to such a truncated site with comparable affinities, indicating that the mutation specifically affects the interaction of IHF with this TTA sequence. This indicates that residue Arg-46 of the wild type HimD subunit, which is conserved in all HimD proteins known so far, is in close contact with the TTA part of the ihf site, and its positive charge might possibly serve to neutralize the negative charge of the phosphates in this DNA region. The arginine residue might also be more directly involved in recognition of the TTA sequence, since for different proteins it has been shown that arginine side chains interact with the minor groove of specific DNA sequences. The PRGRPK motif of the HMGI/Y protein for instance makes with the arginine side chains specific contacts in the minor groove of the AAATTT sequence(30) . The arginine side chains of the N-terminal arm of the engrailed homeodomain (RPRTAFS) make minor groove contacts with the TAAT sequence (31) and the first arginine of the extended polypeptide chain of the DNA binding domain of the Hin protein (GRPRAITKH) is located in the minor groove where it interacts with a specific A-T base pair of the binding site(32) .

In agreement with our results, it has recently been reported that the turn between sheets 1 and 2 of the HimA subunit might be directly involved in contacting the DNA in the 5` non-consensus half of the IHF-binding region of the H` site of phage . This was shown by photocross-linking of this DNA region to a region of the HimA protein from residues 45-54, which comprises the turn region. Interestingly, the turn of the HimA subunit contains a serine residue at the position corresponding to Arg-46 in HimD. This serine is also conserved in all HimA proteins that have been analyzed until now. It is not known whether this serine residue makes direct contacts with the DNA, since residue Gly-49 was indicated as the major site for the cross-linking. If the serine is involved in a direct contact with the DNA, it might be through sequence-independent interactions with the phosphate backbone.

Although the TTR (TTA in the case of the Mu ihf site) part of the ihf consensus forms an important determinant for IHF binding, we have shown that a specific IHF-DNA complex can still be formed when a truncated DNA fragment is used that lacks this sequence, albeit with a much lower affinity. When, however, the 4 nucleotides located between the TATCAA and the TTA part are removed, no specific complex is obtained. Apparently these four nucleotides have an important function in complex formation, probably because they also participate in contacts with the IHF protein. Such contacts would be mediated by the phosphates of the sequence, since comparison of known ihf sequences does not show nucleotide preferences in this region(16) . If the arm of HimA contacts the TATCAA region and the turn between 1 and 2 of HimD contacts the TTA region, then the C-terminal part of HimD with the 3 helix and the 4 amino acids extension would be a good candidate to interact with the sequence in between (Fig. 9). We have shown that deletion of the 4 C-terminal amino acids of HimD have only a minor effect on IHF binding. This is in agreement with the observation that substitution of these 4 residues have no effect on the function of IHF in vivo(10) . The additional deletion of the 3 helix of HimD did reduce the binding affinity, but specific IHF-DNA complexes could still be formed. These properties would fit with a role for the 3 helix in interacting with the DNA that separates the two parts of the consensus, since the arm- and turn-interactions would still allow the formation of the specific complex. The recent observation that the 3 helix of HimD can be functionally replaced by the 3 helix of HU (33) would be consistent with this model. On the other hand, it is very well possible that the 3 helix of HimD has an indirect role in the stability of the IHF-DNA complex through interaction with another part of the IHF protein. This seems certainly to be the case for the 3 helix of the HimA subunit. Deletion of this domain from HimA has a much more drastic effect on IHF, since it completely loses its ability to bind to the ihf site. The affinity for nonspecific DNA however does seem comparable to that of normal IHF. We do not regard it as very likely that these properties reflect a direct function for the 3 helix of HimA in recognizing the consensus sequence. If the 3 helix would contact the DNA, it would be with the DNA region that is located between the sequences that could be cross-linked to the arm of HimD and to the turn-region of HimD (34) (Fig. 9). The sequence of this region does not show a significant similarity score when different ihf sites are compared, indicating that it is not a determinant in ihf site recognition. Although we cannot exclude the possibility that the 3 helix of HimA contributes to the stability of the IHF-DNA complex by interacting with the DNA, its role in the specificity of binding is probably indirect by stabilizing the structure of other DNA binding domains. For the homologous histone-like protein TF1 from Bacillus subtilis, it has been described that the 9-amino acid region that extends beyond the C-terminal 3 helix is important for specificity of SPO1 DNA binding(35) . Recent NMR analysis (36) has indicated that this 9-amino acid tail of TF1 also adopts an -helical conformation. The elongated -helical region of one TF1 subunit shows numerous NOE contacts with the arm domain of the other subunit and with the turn between the 2 helix and the 1 sheet of the other subunit. The HimA subunit also contains a C-terminal extension, but we have shown that this extension does not contribute to the specificity of IHF binding, whereas the 3 helix does. The proposed stabilizing function of this 3 helix could be mediated in two ways. It could either stabilize the structure of the HimD arm through direct interaction with this domain. Since the 3 helix is at the end of the HimA arm, it could also stabilize the structure of this arm through interaction with the turn between 2 and 1 of HimD, thereby anchoring the HimA arm to the body of the protein.

IHF is a heterodimeric protein with two subunits that share only 25% amino acid identity. For the model of the IHF-DNA complex, however, the structure of the symmetrical HU homodimer is used. Although the cross-linking data of Yang and Nash(34) , combined with the binding properties of the R46H mutant that are described in this study, strongly argue for a symmetrical wrapping of the DNA around the IHF molecule with similar domains of each subunit interacting with the DNA, the intrinsic asymmetry of the IHF molecule might be important for the complex formation. The differential effect that we observe after deleting either the 3 helix of HimA or the 3 helix of HimD might be a reflection of such an asymmetry.


FOOTNOTES

*
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.

§
To whom correspondence should be addressed: Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands. Tel.: 71-27-47-73; Fax: 71-27-45-37.

The abbreviations used are: IHF, integration host factor; bp, base pair(s).

W is either T or A, and R is A or G. N represents any of the four nucleotides.

N. Goosen, unpublished data.

L. Zulianello, P. van Ulsen, P. van de Putte, and N. Goosen, unpublished data.

P. van Ulsen, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Kees Franken and Geri Moolenaar for their advice and helpful discussions. We are grateful to Hans den Dulk, Nathalie Chevallier, and Stano Stuchlik for technical assistance.


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