(Received for publication, November 22, 1995; and in revised form, January 25, 1996)
From the
The CI repressor protein, responsible for maintenance of the lysogenic state, and the Apl protein, required for efficient prophage induction, are the two control proteins of the lysis-lysogeny transcriptional switch of coliphage 186. These proteins have been overexpressed, purified, and their self-association behavior examined by sedimentation equilibrium. Phage 186 CI dimers self-associate in solution through tetramers to octamers in a concerted process. The Apl protein of 186 is an unusual example of a helix-turn-helix protein which is monomeric in solution.
Bacteriophage has served as a model system for describing
mechanisms of gene control in both prokaryotes and higher organisms. In
particular, the means by which the
CI and Cro proteins interact
with their operator sites to control transcription, and so foster
either lysogenic or lytic development, has been studied extensively by
a range of genetic, biochemical, and physiochemical approaches. These
studies have contributed enormously to our understanding of genetic
control mechanisms(1) . Bacteriophage 186 from the P2 family of
phages has a completely different nucleotide sequence to
and has
evolved a different set of mechanisms for controlling expression of its
genome. Coliphage 186, like
, is able to replicate its genome
through one of two independent but interchangeable pathways. The lytic
pathway results in lysis of the host cell and production of progeny
phage, while the lysogenic pathway involves integration of the phage
genome into the chromosome of the bacterial host where it is replicated
along with the host chromosome in subsequent generations. The lysogenic
state in 186 is an extremely stable one: the frequency of uninduced
transition from lysogeny to lytic development approaches the mutation
rate(2) . Despite the stability of the lysogenic state, the
correct environmental stimuli can induce 186 to rapidly and efficiently
switch from lysogeny to lytic development, the process of prophage
induction(2) . We anticipate that investigation of the
mechanisms by which this switching occurs will further our
understanding of general genetic control strategies.
The
lysis-lysogeny switch region of 186 (Fig. 1) involves two face
to face promoters, p and p
,
whose transcripts overlap by 62 base pairs (5) . The lysogenic
state is maintained by the product of a single gene, CI, transcribed
from the leftward lysogenic promoter, p
(3) . CI represses transcription from the
rightward lytic promoter, p
(5) as well as
directly repressing transcription of the late control gene B from the p
promoter(8) . In addition,
there are two flanking sites whose function is unknown, one (FL),
located within the cI gene, the other (FR), found at the 5`
end of the apl gene(7) . The Apl protein of 186,
produced from the first gene of the rightward early lytic transcript,
has no apparent role in lytic development after infection but is
required for efficient prophage induction(6) . It functions
both at the level of derepression and of prophage excision(6) . (
)In this regard, Apl assumes the roles of both the Cro and
Xis proteins of phage
. Consistent with its dual functions as
repressor and excisionase, Apl binds between the p
and p
promoters and at the attP site for integrative-excisive recombination (Fig. 1)(6) .
Figure 1:
Organization of the major control
region of 186. The map of the early region of 186 from the PstI site (65.5%) to the BssHII site (76.8%) is
shown(3, 4, 5, 6, 7) .
Sequence numbering begins at the PstI site at 65.5%. Genes are
shown as boxes (rightward genes above the line, leftward genes
below), promoters as arrowheads, their transcripts as arrows, and terminators as stem-loops. p is the lysogenic promoter, p
is the early
lytic promoter, and p
is the promoter for the B gene, the product of which activates transcription of late
genes. cII is the gene required for establishment of lysogeny, int is the integrase, and 69 is of unknown function.
The phage attachment site for integration into the host chromosome, attP (Reed et al.
) is shown. CI binding
sites (7) are indicated by solid boxes, while Apl
binding sites are shown as cross-hatched boxes. The MaeII 2666 to MaeIII 2668 (switch) region is enlarged
in order to present the relative arrangement of the Apl and CI binding
sites. The -10 and -35 regions of the p
and p
promoters are indicated, as are the
start sites for transcription (+1).
We wish to investigate the molecular
mechanisms by which these two proteins, CI and Apl, act to control gene
expression. Any description in quantitative terms of a protein-DNA
interaction must include consideration of each of the equilibria
involved. Since DNA binding proteins rarely act as single structural
units, but tend to exist as dimers or higher oligomers (either
pre-existing or induced upon binding to DNA(9) ), any such
protein self-association must be taken into account. Hence, as a first
step in understanding the molecular mechanisms by which (i) CI is able
to repress transcription from p and p
, and thereby efficiently maintain the lysogenic
state, and (ii) Apl is able to function both as a repressor and as an
excisionase in bringing about lytic development, we have overexpressed
and purified both proteins. Further, we have examined by analytical
ultracentrifugation the ability of CI and Apl to self-associate and
found that in solution CI higher order self-assembly proceeds in a
concerted manner from dimer through tetramer to octamer, while Apl
remains monomeric over the concentration range examined.
Radiolabeled nucleotides, acrylamide solutions, and oligonucleotide primers were purchased from Bresatec (Adelaide), while restriction enzymes were from New England Biolabs. All chemicals were of reagent grade or better.
For expression of CI, BL21 (DE3) plysS pET3aCI cells
were grown at 37 °C in 2-liter flasks containing Luria Broth (500
ml), 100 µg ml
carbenicillin, and 30 µg
ml
chloramphenicol. When the culture had reached an
optical density of 0.6-0.8 at 600 nm,
isopropyl-1-thio-
-D-galactopyranoside was added to a
final concentration of 0.4 mM, and growth was continued for an
additional 3 h. Cells were collected by centrifugation, washed once
with 50 mM Tris-HCl, 0.1 mM EDTA, 10% glycerol, 150
mM NaCl, pH 7.5 (TEG 150) buffer, and stored at -70
°C in approximately 10 ml of the same buffer. Expression of Apl was
the same as that described for CI, except that growth was at 30 °C
in order to maximize the fraction of soluble protein.
Protein concentrations were measured using the Bio-Rad protein assay
or by absorbance at 280 nm. Extinction coefficients were calculated
from the average extinction coefficients of tryptophan (5500 M cm
) and tyrosine (1200 M
cm
), assuming
additivity of absorbances(11) . These values are 23,470 M
cm
for CI and 14,600 M
cm
for Apl.
At sedimentation
equilibrium, the total concentration, c, of a
reversibly self-associating species at radial distance, r, can
be expressed as
where c
Substitution into and rearrangement gives, for i = 2,
where equilibrium constants are fitted as ln K to
constrain them to positive values. Additional terms (e.g. i = 4 for tetramer) can be added to for more
complex association schemes. Hence, on the basis of data obtained from
the ultracentrifuge (total concentration, on an absorbance scale, as a
function of radial distance), equilibrium constants describing a given
association scheme can be obtained by fitting the data to .
These fitted values of K are then converted from an absorbance
scale to a molar scale, based on the degree of polymerization and the
appropriate extinction coefficient, (corrected for the pathlength
of the centerpiece).
Data sets used in the analysis were truncated to include only
absorbance values below 1.2, to ensure absorbance is linear with
respect to protein concentration. Nonideality was not considered. Data
analysis was done using a commercial graphics/curve-fitting program
(Sigmaplot 5.1, Jandel Scientific, Corte Madera, CA) or by
NONLIN(13) . The partial specific volumes of CI and Apl were
calculated using the amino acid partial specific volume values of
Zamyatnin(14) . These calculations gave t;ex2html_html_special_mark_amp;ngr; = 0.725 ml g for CI and t;ex2html_html_special_mark_amp;ngr; = 0.735 ml
g
for Apl. Buffer density at 5 °C was measured
in an Anton-Paar precision density meter to be 1.0378 g
ml
.
SDS-PAGE of samples taken at various stages of the purification of CI are shown in Fig. 2A. In purifying CI, PEI precipitation results in the coprecipitation of CI with nucleic acids. CI is then efficiently separated from the nucleic acids by extracting the PEI pellet with a buffer of moderate salt concentration. Ammonium sulfate precipitation serves to separate CI from any remaining PEI and some of the protein contaminants. CI bound strongly to the Affi-Gel Blue column and was eluted as a broad peak to give a protein pool containing only a few contaminants. These were removed by chromatography on a heparin affinity column. An additional chromatography step on a Superdex 75 HR 10/30 fast protein liquid chromatography gel filtration column showed no evidence of contaminating species (not shown).
Figure 2: SDS-PAGE of aliquots from various stages during the purification of CI and Apl. Samples were run on 15% polyacrylamide-SDS gels. Following electrophoresis, the gels were stained with Coomassie Blue. A, CI purification. Lane a, cleared lysate; b, PEI extract; c, ammonium sulfate precipitation; d, Affi-Gel blue column; e, heparin column; f, molecular mass markers. B, Apl purification. Lane a, cleared lysate; b, PEI/ammonium sulfate precipitation; c, Sephacryl S200; d, molecular weight markers. The sizes of the molecular mass markers are indicated in kilodaltons.
Fig. 2B shows SDS-PAGE of samples taken during the purification of Apl. As was the case for CI purification, PEI precipitation was used to remove nucleic acids and some contaminating proteins. However, Apl did not coprecipitate with the nucleic acids but remained in the supernatant, behavior consistent with its predicted isoelectric point of 10. Ammonium sulfate precipitation was used to concentrate the protein and separate it from PEI for the gel filtration procedure. The small size of the Apl protein (9.6 kDa) allowed purification in a single step on Sephacryl S200, under denaturing conditions. With conservative pooling of Apl-containing fractions, Apl was obtained with >95% purity. Refolding was performed by dialysis against progressively lower concentrations of urea. Since Apl contains only a single cysteine(3) , refolding was straightforward and very little precipitate was observed.
The UV spectra of purified CI and Apl are
typical of those obtained for tryptophan-containing proteins (not
shown). The A/A
ratios
are 1.69 for CI and 1.84 for Apl, indicating the absence of significant
quantities of contaminating nucleic acids.
In order to gain a qualitative estimate of the
extent of any self-association, purified CI and Apl were subjected to
gel filtration chromatography on a column of Sephacryl S200. By
calibrating the column with a series of proteins of known molecular
weight, the elution volume of the protein of interest can be used to
infer an apparent molecular weight. It should be emphasized that for
small zone experiments, the technique is dependent upon the assumption
of spherical geometry and that no simple relationship exists between
protein concentration and elution volume. At a loading concentration of
1 µM, CI eluted from the column with an apparent molecular
weight of 60,000, approximately 3 times that of the monomeric species (Fig. 3). When CI was loaded on the column at a 10-fold higher
concentration (10 µM), the apparent molecular weight
increased to 158,000, approximately 7 times that of monomer. This
concentration dependence of elution volume indicates that CI does
indeed self-associate in solution. On the other hand, Apl eluted with
an elution volume greater than that of the smallest molecular weight
standard (cytochrome c, M =
12,400), indicating that it undergoes little self-association in
solution.
Figure 3:
Small zone gel filtration of purified CI
and Apl on Sephacryl S200. Three hundred-microliter samples were loaded
on a 1 44 cm column of Sephacryl S200 equilibrated with 50
mM Tris-HCl, 0.1 mM EDTA, 100 mM NaCl, 10%
glycerol, pH 8.0. The column was eluted at 0.25 ml min
and elution volumes (V
) were
calculated by monitoring absorbance at 280 nm. Protein standards used
to calibrate the column (solid symbols) were amylase (200
kDa), alcohol dehydrogenase (150 kDa), carbonic anhydrase (29 kDa), and
cytochrome c (12.4 kDa). The void volume (V
) was taken as the elution volume of blue
dextran (Pharmacia). Open symbols indicate the elution
positions of CI when loaded on the column at 10 µM (a) and 1 µM (b), while the arrow (c) shows the elution position of Apl protein (loading
concentration 16 µM). The broken line indicates
that this part of the plot is outside the range covered by the protein
standards.
The ability of 186 CI and Apl to self-associate in solution was investigated in a quantitative manner by sedimentation equilibrium. All experiments were performed at 5 °C in TEG 150 buffer. For both proteins, different combinations of loading concentration and rotor speed were used. Fig. 4shows the results of three of the sedimentation equilibrium experiments performed on CI with a loading concentration of 9 µM (in terms of total repressor subunits) at rotor speeds of 12,000, 16,000 and 24,000 rpm. Initially, the individual concentration distributions were fitted to the equation for a single species (i = 1) in order to obtain whole cell average molecular weights. These ranged from 91,000 (±1000) to 144,000 (±2300). Thus, the greatest whole cell average molecular weight is approximately 6.8 times that of the monomeric species. These results confirm that CI self-associates and that the self-association is to species larger than hexamer.
Figure 4: Sedimentation equilibrium of 186 CI at 5 °C. Experiments were performed at 12,000 rpm (open squares), 20,000 rpm (solid circles), and 24,000 rpm (open circles). The data are shown as the concentration distribution as a function of radial distance. The lines represent the best fit of the data to a dimer- tetramer-octamer association scheme, while the lower residuals plot shows the difference between the experimental and fitted values. For clarity, only every third data point is shown.
The
sedimentation data sets were then analyzed globally in terms of various
assembly schemes. Insufficient data could be obtained at the low
concentration end (even when distributions were recorded at 230 nm) to
satisfactorily describe the monomer-dimer interaction and so, in
subsequent analyses, the smallest species in the association scheme was
set at that of a dimer (M = 42,320). When
constraining M
() to this value, the best fit
to the data, as judged by the sum of squares of residuals (SSR), was to
a dimer-tetramer-octamer equilibrium (
G
= -7.0 ± 0.1 kcal mol
,
G
= -21.3 ± 0.1 kcal
mol
, SSR = 0.007). Consistent with this
result, when the molecular weight of the smallest species
(M
) was allowed to float in the calculation, essentially
the same free energies of association were obtained and the resulting
fitted value for M
was, within experimental error, that of
a CI dimer (43,390 ± 1340). Including additional species in the
association scheme (dimer-tetramer-hexamer-octamer) did not improve the
fit above that expected solely on the basis of fitting to an additional
parameter.
Short pathlength cells (2.5 mm) were used with a higher
loading concentration of CI (20 µM) in order to better
define the high end of the association scheme. However, inclusion of
these data sets into the global fit did not justify inclusion of
additional species larger than octamer. Fitting the data sets to a
dimer-octamer equilibrium (i.e. no formation of tetramer, K = 0) resulted in a fit similar to that
obtained for the dimer-tetramer-octamer scheme, reflecting the
difficulty in defining the association constant for species which do
not accumulate to a significant degree. Hence, the self-association of
the 186 CI repressor protein under the conditions studied is best
described by a dimer-tetramer-octamer equilibrium, the dimer-octamer
transition being a concerted process (see ``Discussion'').
Apl self-association was also studied by sedimentation equilibrium. Four runs were performed, employing two different rotor speeds (16,000 rpm and 24,000 rpm) and two different loading concentrations (16 µM and 32 µM) (Fig. 5). Data sets from all four runs were fitted globally to various self-association schemes. The best fit to the data was that of a single monomeric species. The fitted value of molecular weight was 10,410 ± 60 (SSR = 0.041).
Figure 5: Sedimentation equilibrium of 186 Apl at 5 °C. Experiments were performed at 16,000 rpm (solid symbols) and 24,000 rpm (open symbols) with loading concentrations of 16 µM (circles) or 32 µM (squares). The data are presented as concentration distributions as a function of radial distance. The lines represent the best fit of the data according to a single monomeric species, while the lower residuals plot presents the difference between the experimental data and the fitted values. For clarity, only every third data point is shown.
Two of the major control proteins from bacteriophage 186, CI
and Apl, have been expressed, purified, and their self-association
properties examined. CI reversibly self-associates in solution, and
this self-assembly is best described by a
monomer-dimer-tetramer-octamer equilibrium. Fig. 6shows the
distribution of CI species calculated from the free energies of
association obtained from fitting the sedimentation equilibrium data to
a dimer-tetramer-octamer association scheme. Even at the lowest
concentration of CI used in the sedimentation equilibrium experiments,
there was insufficient monomer present to characterize the
monomer-dimer equilibrium. In order to permit inclusion of the
calculated distribution of monomer and dimer in Fig. 6, a value
of K has been estimated. This estimate, 1
10
M
(
G
= -10.2 kcal
mol
), is based on the detection limit of the
ultracentrifuge; that is, any value of K
lower
(weaker) than 1
10
M
would have been resolved in the fitting procedure. Based on this
value of K
, CI exists primarily as a mixture of
monomers (21 kDa) and dimers (42 kDa) between 10
and 10
M (in terms of monomer). Below
10
M, CI is essentially monomeric. Use of
a 10-fold higher value of K
results in the
monomer-dimer curves shifting one log unit to the left, without
significant change to the tetramer and octamer curves (not shown).
Between 10
and 10
M, CI
exists in solution as a mixture of dimer (42 kDa), tetramer (84 kDa),
and octamer (168 kDa), with octamer being the predominant species at
concentrations above 10 µM. The tetrameric species exists
only as an intermediate during the assembly of dimers to octamers,
never reaching more than 35% of the total. The distribution of tetramer
is subject to some uncertainty given the difficulty in precisely
defining K
. Within the concentration range
examined, there was no evidence for formation of polymers higher than
octamers. That octamer formation is a concerted (favored) process can
be seen by calculating the free energy per dimer required for formation
of the higher species, for tetramer formation,
G
= -3.5 kcal mol
per dimer, while
for octamer formation,
G
= -5.3
kcal mol
per dimer. Thus, the free energy per dimer
for octamer formation is more negative than the free energy per dimer
of tetramer formation, and assembly of dimers to octamer is the
energetically favored process.
Figure 6:
Distribution of CI oligomeric species as a
function of total CI concentration, in total repressor subunits. The
weight fraction of each species present was calculated using the free
energies of association obtained from the sedimentation equilibrium
experiments (G
= -7.0 kcal
mol
,
G
=
-21.3 kcal mol
). A value of -10.2 kcal
mol
was used for the monomer-dimer equilibrium
(
G
, see
``Discussion'').
Like 186 CI, the CI repressor
also associates from dimer through tetramer to octamer in a concerted
process(16) , although at very high protein concentrations (up
to 100 µM) the
data were consistent with a further
association to dodecamer. Not only do the
and 186 repressors
assemble in solution in the same manner, but comparison of the fitted
free energy values for the various steps in the association process
shows a remarkable similarity. For the dimer-tetramer equilibrium,
G
is -7.1 kcal mol
for
and -7.0 kcal mol
for 186,
while the free energies for the dimer-octamer equilibria are also quite
similar;
G
= -23.0 kcal
mol
for
CI and -21.3 kcal
mol
for 186 CI. Given this correspondence between
the self-association characteristics of the two proteins, one might
expect their structures to show some similarity. While the domain
structure of the
repressor has been studied extensively, little
is known about the tertiary structure of 186 CI. Several lines of
evidence suggest that
CI consists of two domains (17) .
The N-terminal domain is responsible for DNA binding and the C-terminal
domain contains the determinants for oligomerization and cooperativity.
The N domain interacts with DNA via a helix-turn-helix motif, a motif
common to many DNA-binding proteins. In the 186 CI repressor, a region
in the N-terminal third of the amino acid sequence gives a weak match
to a helix-turn-helix motif as judged by weight matrix
analysis(18) . We suspect that 186 CI contains a variant form
of this DNA binding motif(7) . Other than this, there is little
similarity at the nucleotide or amino acid level between the
and
186 repressors. The nature of the operator sites to which the two
repressors bind do share some features. The repressors bind to three
operators in both
at O
(O
1,
O
2, and O
3) and 186 at p
(site II, site I, and site III), each operator being separated by
approximately two turns of the helix(1, 7) . In 186,
however, the central operator (site I) has a consensus sequence
unrelated to that of the two adjacent operators, indicating that 186 CI
may contain more than one region capable of binding DNA (7) .
DNase I footprint analysis of mutated 186 p
operator regions show evidence of cooperativity(7) .
What are the implications of these results for 186 CI binding to its
operator sites? Protein-protein association in solution to provide
multidentate ligands capable of binding multiple sites on DNA is a
common mechanism for cooperative binding of regulatory proteins to DNA (16) . Thus, in order to fully characterize a protein-DNA
interaction, oligomerization of the protein must be considered. For
example, as discussed by Senear et al.(16) , linkage
between protein self-assembly and DNA binding may produce free energy
changes for oligomerization which will differ depending on whether
binding to DNA favors or disfavors protein self-assembly. In the case
of , Laue et al.(19) found that binding of
O
1 oligonucleotides to octameric CI repressor did not
dissociate it to tetramers, and, therefore, any model for
which
proposes pairwise cooperativity between adjacent DNA-bound dimers (to
give DNA-bound tetramers) must also consider the free energy required
to destabilize the octamer. Similarly, in the case of 186, further
studies of the protein-protein and protein-DNA interactions are
required to delineate the contributions of cooperativity, linkage, and
allostery to the molecular mechanism by which CI binds to its operator
sites and stably maintains the lysogenic state.
In considering the
ability of CI to maintain the lysogenic state, one must also be aware
of the relative arrangement of the lytic and lysogenic promoters (Fig. 1). An inevitable consequence of the overlapping face to
face arrangement of the p and p
operators in 186 is that RNA polymerase, in transcribing CI from p
, must traverse p
in order
to maintain the lysogenic state. In doing so it must presumably
dislodge CI already bound at p
, providing the
opportunity for loss of repression. A possible mechanism for preventing
this loss of repression involves the flanking sites FL and FR providing
a locally high concentration of CI, allowing rapid rebinding of CI to p
following passage of RNA polymerase. Such a
mechanism would require oligomerization of CI such that it could bind
simultaneously to p
and either FL or FR. These
studies have demonstrated that this oligomerization can occur, at least
in solution.
Turning now to Apl, this protein functions as a
repressor during prophage induction and is involved in excision of the
prophage from the bacterial host, roles performed by two proteins, Cro
and Xis, in phage . Apl binds to a set of seven direct repeats at p
/p
and five direct repeats
at the attachment site, attP (Fig. 1; (5) ).
These sites have 10-11-base pair center to center spacing,
indicating that Apl binds to the same face of the helix. Given the
narrow range of concentration over which Apl fills the multiple
operator sites within the p
/p
region(6) , cooperative interactions must be involved.
Apl, like the homologous Cox proteins from P2 and HP1 phage, has a
predicted helix-turn-helix DNA binding motif(18, 20) .
In general, helix-turn-helix proteins are dimers or tetramers in
solution (for example, the majority of those listed in(21) )
and it is these oligomers which interact with their operator sites,
usually (but not always) inverted repeat sequences. In contrast,
analytical ultracentrifugation of purified Apl shows that Apl remains
monomeric in solution up to millimolar concentrations.
It could be
argued that since Apl has been denatured and refolded during the course
of the purification, the majority of the protein is in an inactive,
nonassociating form and that the activity observed in gel shift assays
arises from a small fraction of active associated protein. While we
cannot completely rule this out, three lines of evidence argue against
this possibility. Firstly, Apl purified to approximately 80% purity by
ion exchange chromatography (without unfolding) eluted on a calibrated
Sephacryl S200 column (Fig. 3) with the same elution volume as
Apl purified by the unfolding/refolding procedure. This indicates that
the ``native'' protein is the same size as the protein
purified by unfolding/refolding. Secondly, this partially purified Apl
fraction had approximately the same specific activity in gel shift
assays as the pure refolded Apl. The yield and extent of purification
of Apl (Table 2) is consistent with retention of DNA binding
activity throughout the purification procedure. Finally, while
monomeric helix-turn-helix DNA binding proteins are unusual, there are
examples in the literature. Thus, while Cro is a dimer in
solution, Cro protein from phage 434 remains monomeric, even at the
high concentrations employed for crystallization(22) . Again,
the biotin operon repressor (BirA) remains monomeric at concentrations
2-3 orders of magnitude higher than the concentration required
for operator binding(23) . Of particular interest are the Ner
proteins of bacteriophage Mu and the closely related phage D108. Like apl, ner is functionally analogous to
cro in that it is the first gene encoded by the early lytic operon and
that its protein product binds between the lytic and lysogenic
promoters to negatively regulate transcription(24) . The
overlapping, face to face arrangements of the lytic and lysogenic
promoters of Mu and D108 are quite similar to 186. Like Apl, Ner binds
symmetrically between the two promoters(25, 26) . Mu
Ner binds, with a dissociation constant in the nanomolar range, as a
tetramer on a 30-bp oligonucleotide(26) , yet is monomeric in
solution at a concentration of 25 µM(27) .
Similarly, D108 Ner forms dimers on DNA but is monomeric in solution up
to 200 µM(28) . Although the tertiary structures
of the Ner proteins are not known, they are selected by the weight
matrix method of Dodd and Egan (18) as being potential
helix-turn-helix proteins, albeit with a relatively weak score.
Given the monomeric nature of the Apl protein in solution and the
fact that Apl is expected to bind cooperatively to its recognition
sequences, it follows that this cooperativity can only be mediated on
the DNA. This is illustrated in Fig. 7which shows a simple
thermodynamic cycle for a protein (A) binding to multiple
sites on DNA. The protein-DNA complex can be formed in two ways. The
protein can either self-associate in solution and then bind to DNA (KK
pathway) or can bind as
monomers to the DNA (K
K
pathway) where the protein subunits may or may not interact to
give rise to cooperativity. A combination of these pathways is also
possible, if both the monomeric and the self-associated forms of the
protein have affinity for the DNA. The overall equilibrium will then
reflect the relative affinities of the two forms of the protein for the
DNA. For Apl, K
, the protein self-association
constant in solution is 0. Apl binding therefore can only occur through
the K
K
pathway: DNA binding
followed by protein-protein association. The structural basis of
cooperativity in Apl binding is unknown, but, given the periodic
enhancements of DNaseI cleavage noted in footprint
experiments(6) , we speculate that it involves both
protein-protein contacts and DNA bending.
Figure 7:
Thermodynamic cycle for a self-associating
protein (A) binding to DNA. Shaded areas represent
DNA binding sites. According to this scheme, the protein can either
self-associate in solution (K) followed by binding
of the oligomeric form (K
) or the monomers can
bind directly to DNA (K
), followed by
oligomerization on the DNA (K
). In the case of
Apl, the protein self-association constant, K
, is
0 and so binding to DNA must occur via the K
K
pathway.
The present results have provided a framework on which to base models describing the interaction of these control proteins with their operators in the 186 control region. We are currently undertaking further studies of both CI-DNA interactions and Apl-DNA interactions in order to dissect at the molecular and energetic levels the mechanisms by which these proteins control the lysis/lysogeny switch in bacteriophage 186.