(Received for publication, May 15, 1995; and in revised form, July 11, 1995)
From the e 1, D-40225
Düsseldorf, Federal Republic of Germany
Here we report a structural investigation of the transcription
factor H-NS and its DNA interaction. H-NS has a general effect on
transcription by compacting DNA; but for a number of specific genes, it
is known to act directly as repressor or activator. The homodimeric
protein binds to the major groove of DNA in a sequence-nonspecific
manner, recognizing a curved conformation of the target DNA. H-NS
consists of 136 amino acids with a single tryptophanyl residue at
position 108. To overcome the apparent lack of any other structural
details, we took advantage of the intrinsic fluorescence of Trp-108.
Static and dynamic quenching constants obtained with the neutral
quencher molecule acrylamide are consistent with a hydrophilic
environment and high degree of solvent exposure for Trp-108. In
addition, quenching studies in the presence of the anionic quencher
iodide indicate a positively charged microenvironment for the same
amino acid residue. Specific and nonspecific H-NSDNA complexes
were studied by gel retardation and fluorescence analysis. While
specific H-NS
DNA complex formation is accompanied by a clear
enhancement of the tryptophanyl fluorescence intensity, interaction in
the presence of the nonspecific competitor DNA poly(dI-dC) decreases
the fluorescence quantum yield.
The Escherichia coli protein H-NS has originally been described as a histone-like, nucleoid-associated protein, considered to be important for cellular mechanisms like DNA compaction or alteration of DNA topology(1, 2, 3, 4, 5) . More recently, it became clear that H-NS acts as an pleiotropic transcription factor involved in the regulation of several unrelated genes(6, 7, 8, 9, 10) . As shown for some other transcription factors, H-NS is known to act as both activator and repressor, depending on the respective transcription unit it controls(6) .
In line with the high binding preference to curved DNA(5, 7, 11, 12) , H-NS-specific regulatory sequences have all been shown to contain a defined intrinsic curvature. Among the genes regulated by H-NS are those involved in osmoregulation and response to environmental stress conditions(8, 13, 14) . In addition, the thermo-osmotic regulation of virulence gene expression in Shigella was shown to be influenced by H-NS(15) . A common denominator for H-NS-dependent regulation of the unrelated group of genes appears to be certain extreme environmental conditions, i.e. cold shock, osmotic shock, or other kinds of stress situations.
The constantly growing number of genes whose expression is known to be affected by H-NS includes the most efficiently expressed RNAs of the cell, namely ribosomal RNAs(16) . H-NS has been shown to act as a specific repressor of the ribosomal rrnB P1 promoter, thereby antagonizing the stable RNA transcription activator Fis under conditions of stationary growth(9, 17, 18, 19) . The site of H-NS/P1 DNA interaction has been mapped for the rrnB promoter by high resolution footprinting techniques. The binding region extends from positions -18 to -89, relative to the P1 transcription start site, and shows an overlap with the known binding sites for Fis. Thus, in addition to the control of several other global regulatory networks, i.e. stringent or growth rate control, ribosomal RNA synthesis is linked to certain cellular stress conditions via H-NS-mediated repression(9, 16) .
H-NS has a molecular mass of 15.5 kDa and consists of 136 amino acids (1, 2) . Specific binding of the homodimeric protein is known to require bent DNA in a precise orientation with respect to the regulated promoters. Hence, the binding is DNA conformation-specific with no known sequence specificity(9, 20) .
Due to a lack of detailed
structural information on H-NS (for instance, three-dimensional
crystallographic data or high resolution NMR spectroscopy), ()we took advantage of the intrinsic fluorescence of a
single tryptophanyl residue at position 108, which we used as a
structural indicator. The microenvironment of the fluorophor was
investigated by fluorescence quenching studies. Furthermore,
steady-state fluorescence measurements were performed to analyze the
protein conformation in the free and DNA-bound states. The specific
interaction of H-NS with a curved rrnB P1 promoter fragment
was followed by fluorescence measurements and compared with the
nonspecific interaction of H-NS with the competitor DNA poly(dI-dC).
These studies demonstrate that the single H-NS tryptophanyl residue at
position 108 is strongly affected upon DNA binding. The fluorescence
emission intensity is enhanced after binding to specific DNA target
sites. In contrast, the interaction of H-NS with nonspecific DNA
decreases the Trp-108 fluorescence. These data, in correlation with
acrylamide and iodide quenching experiments, suggest that H-NS shows
two different complex conformations dependent on binding to specific or
nonspecific DNA sites and closely links Trp-108 to this conformational
change.
The classical Stern-Volmer equation relates the drop in fluorescence to the concentration of a collisional quencher as shown in :
where F is the fluorescence in the absence
and F is the fluorescence in the presence of the quencher (Q). K
is the Stern-Volmer constant for
the collisional quenching process(24) . According to the
fluorescence quenching data obtained from acrylamide quenching, the
following modified Stern-Volmer equation was used to estimate the
relevant quenching constants:
where now K is the dynamic quenching
constant and V is the additional static quenching constant (24) . To determine K
and V, the
collected data were fitted to using the nonlinear
least-squares program ``FITLS32.'' (
)
Figure 1:
Stern-Volmer plot of the acrylamide
quenching of H-NS fluorescence emission in the presence of 0, 0.025,
0.05, 0.075, 0.1, 0.125, 0.15, 0.175, and 0.2 M acrylamide
(). The inset shows complete H-NS fluorescence emission
spectra in the presence of 0 and 0.2 M acrylamide recorded
between 300 and 400 nm. To obtain a maximum of graphical clearness,
fluorescence emission spectra at 337 nm with different acrylamide
concentrations (indicated above) are shown as crossedcircles.
= 282 nm, 21
°C.
Figure 2:
Stern-Volmer plot of the iodide quenching
of H-NS fluorescence emission in the presence of 0, 10, 20, 30, 40, and
50 mM KI ([trio). The inset shows complete H-NS
fluorescence emission spectra in the presence of 0 and 50 mM KI recorded between 300 and 400 nm. To obtain a maximum of
graphical clearness, fluorescence emission spectra at 337 nm with
different KI concentrations (indicated above) are shown as crossedcircles. = 282 nm, 21
°C.
The
Stern-Volmer plots of the collected acrylamide and iodide quenching
data of H-NS show opposite tendencies: the Stern-Volmer plot of H-NS in
the presence of acrylamide follows an upward curved exponential
function, which can be fitted according to . The acrylamide
quenching data yield apparent static and dynamic quenching constants of V = 4.3 M and K
= 11.7 M
,
respectively. In contrast, the ratio F
/F of the H-NS I
quenching experiments exhibits a
downward curved exponential saturation as shown in Fig. 2. It
can be described with an exponential function such that F
/F = a - (a - 1)
e
. The
downward curvature of such a dependence in quenching experiments can be
explained with the hypothesis that a net positive charge exists in the
microenvironment of the tryptophanyl residue, for example, due to
protonation of nearby amino acid side chains. After saturation of this
positive charge with increasing amounts of iodide anions, following
quenching, interactions with the fluorophor can only occur in a weaker
manner(24) . As we shall see, the notion of a positively
charged microenvironment of Trp-108 is essential for the considerations
below.
Figure 3:
Gel
mobility shift analysis of H-NSrrnB P1 complexes in the
presence of different amounts of nonspecific competitor DNA. Lanes
1-10, 0, 1.2, 2.3, 4.7, 9.3, 14, 18.7, 23.3, 29.1, and 35
µM bp of poly(dI-dC). H-NS
indicates a supershift of P1 DNA caused by additional
nonspecific (unspecific) interactions of H-NS with the promoter
fragment. H-NS
shows specific
DNA
protein complexes. P1
indicates unbound DNA.
Figure 4:
Fluorescence emission spectra of H-NS in
50 mM Tris-HCl, pH 7.4, in the presence of a constant 35
µM bp of competitor DNA poly(dI-dC) and different amounts
of rrnB P1 UAS DNA. Protein concentration was 86 nM.
= 282 nm, 21
°C.
Figure 5:
Fluorescence emission spectra of H-NS in
50 mM Tris-HCl, pH 7.4, in the presence of a constant 35
µM bp of competitor DNA poly(dI-dC) and different amounts
of additional poly(dI-dC) DNA at the same concentrations as the P1 UAS
fragment shown in Fig. 4. Protein concentration was 86
nM. = 282 nm, 21
°C.
A typical H-NS fluorescence emission spectrum between 300 and 400 nm can be recorded when the control samples without the P1 fragment or additional poly(dI-dC) are excited at 282 nm. The addition of the specific binding P1 UAS fragment results in a clear increase in fluorescence intensity, indicative of a change in the environment of the fluorophor, caused either by an alteration of the protein conformation or by the close proximity of the DNA (Fig. 4). In contrast, the addition of nonspecific DNA under the same conditions leads to a decrease in the tryptophanyl fluorescence emission intensity (Fig. 5). Thus, the Trp-108 environment of H-NS after complex formation with specific or nonspecific DNA is different, indicating conformational differences in H-NS or differently arranged complex topographies.
Apart from its early
characterization(1, 22) , little is known about the
structure and mode of DNA interaction of the transcription regulator
H-NS. The neutral chromatin-associated protein shows a rather
nonspecific affinity for nucleic acids including double-stranded DNA of
random sequence, single-stranded DNA, or even RNA(27) .
Preferential binding of H-NS with much higher affinity and specificity
has been demonstrated to intrinsically curved
DNA(8, 9, 10, 12) . In those cases,
the protein apparently acts as a specific transcription factor.
However, no common DNA sequence motif for specific binding is known,
and the available evidence indicates that interaction depends
exclusively on specific DNA conformations
(curvature)(9, 12, 20) . This unusual binding
property is reflected in the complete absence of peptide motifs
commonly found in DNA-binding proteins, i.e. helix-turn-helix,
zinc fingers, or other DNA-binding elements. To understand the binding
mechanism, structural information about the protein and
proteinDNA complexes is required. Because the protein contains a
single tryptophan residue, we performed fluorescence analysis as the
method of choice.
Fluorescence quenching studies have provided
valuable information concerning the exposure of the tryptophanyl
residues of proteins in the past(24) . Comparison with
quenching data on other proteins helps to determine the degree of
exposure of the fluorophor. The magnitude of the static and dynamic
acrylamide quenching constants (V = 4.3 M and K
= 11.7 M
) obtained for H-NS indicates that the
microenvironment of the H-NS tryptophanyl residue at position 108 is
totally exposed to the solvent. For instance, in the case of the
galactose repressor protein GalR (V = 0.3 M
and K
= 5.3 M
), Brown et al.(28) postulated some steric shielding of the tryptophan
residue. In this case, the microenvironment is not totally protected
from the solvent(28) . In contrast, the quenching constants for
glucagon (V = 1.0 M
and K
= 10.5 M
)
are greater than those found for GalR. Here, the tryptophan residue of
glucagon is considered to be solvent-exposed(29) . The
magnitude of the quenching values for H-NS presented in this study are
similar or greater than the values for glucagon, indicating that the
tryptophanyl residue of H-NS is, as in glucagon, totally exposed to the
solvent.
The Stern-Volmer plot of the iodide quenching experiments exhibits a downward curved exponential saturation. Because both the fluorescence acrylamide and iodide quenching studies of H-NS show a significant drop in fluorescence emission intensity without spectral shift and since DNA-binding proteins are almost always symmetrical multimers, we do not favor the possible explanation that the tryptophan residues of one H-NS dimer are in different environments. Rather, we want to point out the more likely fact that the saturational function is due to the circumstance that a net positive charge exists in the microenvironment of the tryptophanyl residue because of the protonation of nearby amino acid side chains. After neutralization of this positive charge with increasing amounts of iodide anions during quenching, interactions with the fluorophor become progressively weaker(24) . This explanation is in good agreement with the high degree of tryptophan solvent exposure demonstrated by the acrylamide quenching results. It is obvious that a region with such positive charge either must be shielded intramolecularly through other negatively charged groups or must be exposed to the environmental medium. The apparently positive charge of the microenvironment of the H-NS tryptophanyl residue at position 108 and its high degree of solvent exposure offer the first indications of a possible interaction during binding of negatively charged DNA to this protein domain.
It is known that at sufficiently high concentrations, H-NS interacts with any double-stranded DNA(30) , which explains its function in the compaction of DNA and changing DNA topology(2, 4) . Strong and specific binding, however, requires DNA with an intrinsic curvature(11, 12) . In accordance, genes under direct repression or activation of H-NS exhibit curved DNA target sequences, to which the protein is known to bind(9, 13, 14) . Specific binding can be prevented in the presence of the drug distamycin, which is known to bind to the minor groove of AT tracts, thereby straightening out curvature(9, 11, 31) . For instance, strong and specific binding to H-NS has been demonstrated for the curved upstream sequence of the ribosomal RNA P1 promoter region, where the interacting sequences have been mapped by footprinting(9) . Therefore, we have studied protein DNA interaction with the rrnB P1 UAS region as an example of strong DNA binding in comparison to the weak interaction of H-NS with nonspecific synthetic DNA poly(dI-dC). Nevertheless, at a high excess of H-NS and in the absence of competitor, nonspecific binding can also be observed to the P1 UAS DNA after all specific sites have been saturated (see, for instance, Fig. 3, lane1).
Studies on the
salt-dependent binding of H-NS to the rrnB P1 promoter
fragment confirm previous findings that H-NS is involved in the
transcription regulation of several important systems. Under high
competitor concentrations, stable binding occurs between 50 and 125
mM KCl, consistent with stable interactions under in vivo conditions, where the intercellular K concentration is about 100 mM(26) . At KCl
concentrations above 130-150 mM, a typical loss of
binding affinity by a factor of 2 can be noticed (data not shown). This
modulation, produced by differences in ionic strength, may in part
underline the linkage of H-NS activity changes with its regulatory
implications under osmotic shock or stationary phase conditions.
In this paper, we describe for the first time molecular details of two distinguishable H-NS binding states depending on the interaction with specific or nonspecific DNA target sites. In the presence of 35 µM bp of competitor DNA, the addition of P1 DNA increases the fluorescence emission intensity dramatically. Under these conditions, the fluorescence results indicate that there must be a specific complex structure that is accompanied by a steric shielding of the tryptophan residue. The increase in the fluorescence emission intensity upon P1 DNA binding indicates more than simple electrostatic interactions and is very likely due to a hydrophobic contact of Trp-108 with staggered base pairs of the DNA major groove. The results do not imply intercalation of Trp-108 into the DNA double strand since this would apparently cause quenching rather than enhancement of the fluorescence intensity. An alternative explanation could be a drastic protein conformational change, shielding Trp-108 and simultaneously exposing domains for specific DNA binding. Both events may not be mutually exclusive.
A possible scenario explaining our data could be as follows. Binding of the protein to the specific P1 target site involves interaction of the positively charged tryptophan microenvironment with the negatively charged DNA phosphate backbone. Thus, the tryptophan residue gets closer to the apolar base pairs and is shielded from solvent influence, which results in an increase in fluorescence emission. This process occurs only if the binding partner of H-NS is a specific DNA ligand and if the salt conditions are suitable for this specific protein/DNA interaction. Obviously, a stringent prerequisite for this specific contact is an intrinsic curvature or high flexibility of the target DNA. It is conceivable that only appropriately curved DNA is able to provide the steric requirements necessary for a close interaction with the H-NS binding domain, which very likely includes tryptophan 108.
In contrast, if
the target DNA does not adopt the right conformation due to the lack of
curvature or bendability, only weak, almost exclusively electrostatic
interactions can be performed. Nonspecific binding is therefore much
more prone to increase in ionic strength or competition by
heparin. Apparently, under such conditions, the H-NS
tryptophanyl residues are more exposed to collisional quencher
molecules from the environmental solvent, which causes a drop in
fluorescence intensity.
In summary, Trp-108 is a suitable sensor responding to specific and nonspecific binding conditions. It is thus conceivable to assume that Trp-108 constitutes part of the H-NS/DNA binding domain.