(Received for publication, April 17, 1995; and in revised form, May 30, 1995)
From the
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 We
also examined the properties of an IHF mutant that has an amino acid
substitution between Integration host factor (IHF) 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 From the binding studies with homodimers of HimA
and HimD (23, 24) and with mutant IHF
molecules(2, 25) 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
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, r
The HimA The HimD-R46H mutant (Fig. 1)
was obtained from a collection of randomly isolated IHF mutants.
Figure 1:
C-terminal parts of different HU and
IHF subunits. A, alignment of the
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.
The HimA 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 HimA
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.
Figure 3:
Tricine protein gel of the purified
proteins. 1, HimA-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
HimA
Figure 4:
Autoradiograph of retarded protein-DNA
complexes with HimA
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
The binding capacity of the mutant IHF
protein that lacks the last
Figure 5:
Autoradiograph of retarded protein-DNA
complexes with the HimA
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 HimA
Figure 6:
Autoradiograph of retarded protein-DNA
complexes with the HimA-HimD
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 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 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 In
agreement with our results, it has recently been reported that the turn
between 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 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
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
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.
(
)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) .
-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.
(
)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) .
sheets 1 and
2 interacts with the TTR part of the consensus and that this
interaction is important for the bending of the DNA.
Strains and Media
Strains AB1157, PP1953
(AB1157, himD), and PP1954 (AB1157, himA) have been
described previously(26) .M
,
himA::Tet) and PP2896 (ompT, r
M
, 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, r
M
,
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) .8- and
HimD
4-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 HimA
15- and HimD
10-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.
(
)For the construction of the R46H-overproducing
plasmid, the NdeI-BamHI fragment containing himD with the corresponding mutation was inserted in
pGP1149(23) .
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.
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.8-HimD dimer was purified by mixing the
lysates from PP2895 with pGP1311 and with pPG1149; the HimA-HimD
4
heterodimeric protein was purified by mixing the lysates from PP2896
with pGP1138 and with pGP1312; in the same way, the HimA
15-HimD
protein was purified from mixed lysates from PP2895 containing the
plasmids pGP1309 and pGP1149, while HimA-HimD
10 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.
8-HimD and HimA-HimD
4 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).
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.
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: HimA
8-HimD and HimA-HimD
4 (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.
4; 2, HimA-HimD
10; 3, wild type IHF; 4, R46H; 5,
HimA
8-HimD; 6,
HimA
15-HimD.
8-HimD and HimA-HimD
4 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
(HimA
8-HimD
4), 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.
8-HimD (A) and HimA-HimD
4 (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 HimA
15-HimD
heterodimer (Fig. 3).
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.
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.
15-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.
15-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-HimD
10 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.
10 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.
-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) .
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.
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) .
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.
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.
3 helix of HimA or the
3 helix of HimD
might be a reflection of such an asymmetry.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.