(Received for publication, September 16, 1994; and in revised form, October 9, 1994)
From the
The sequence of the bacteriophage 434 O1
(ACAAAACTTTCTTGT) differs from its O
3 (ACAGTTTTTCTTGT) at
positions 4-6. X-ray analysis shows that the side chain of
Gln
of the 434 repressor makes van der Waals' and
H-bond contacts with the T at position 4` in complex with
O
1, but no specific contact is observed at this position in
434 repressor-O
3 complexes. No contacts are made by
repressor to the bases at positions 5 or 6 in either binding site. The
significance of the sequence differences between O
1 and
O
3 in determining the operator affinity for repressor were
examined by constructing synthetic variants of these operators.
Measurements of the affinity of these operators for repressor as a
function of ionic strength revealed that although base pairs 5
and 6 are not contacted by 434 repressor, they can nonetheless
influence operator affinity for repressor by modulating the degree to
which ionic interactions contribute to the overall binding energy. Both
the magnitude and direction of their effect depends on the status of
repressor's contacts to the bases at position 4. The role of
contact made by Gln
to position 4 was examined by mutating
this amino acid to Ala and by examining the affinity of wild type
repressor for an operator bearing a 5-methylcytosine at position 4` in
an O
1-4G mutant. These experiments showed that
repressor's preferences at operator positions 5 and 6 are linked
to its position 4 preference via a van der Waals' contact between
amino acid 33 and a methyl group on the base at operator position 4`.
Together, the results of the experiments shown here reveal that bases
that do not contact the protein alter its preferences for bases at the
contacted operator position 4.
Repressor is a DNA binding protein encoded by the bacteriophage
434. Transcription of the genes that define the developmental fate of
the phage in Escherichia coli is controlled, in part, by the
binding of 434 repressor to six binding sites on the bacteriophage
chromosome. These six binding sites are divided between two operator
regions termed O and O
. Repressor's
discrimination between two of these binding sites, O
1 and
O
3, provides a critical hinge point in determining the
transcriptional activity at O
and, therefore, the fate of
the phage in the cell(1, 2) .
Similar to the
repressors of other lambdoid phages (1, 2) 434
repressor protein binds to DNA as a dimer. Each monomer of the protein
is divided into two functionally distinct domains(3) . The
carboxyl-terminal domain (amino acids 71-209) is responsible for
dimerization of the intact repressor, while within R1-69, the
amino-terminal domain (amino acids 1-69) residues in a
helix-turn-helix motif determine DNA binding specificity(4) . Fig. 1shows the sequences of O1 and O
3
and two position 4 variants of those operators. X-ray structural
investigations show that the 434 repressor only makes specific contacts
to the symmetrically arrayed outer four base pairs (positions 1-4
and 11-14 in Fig. 1) in these pseudosymmetric binding
sites(5, 6) . Elimination of these contacts by
mutation at these bases abolishes specific complex
formation(7, 8) . Similar to the case with
repressor(9) , changes in the central 6 bases in the 434
operator (5-10 in Fig. 1) dramatically alter operator
affinity for 434 repressor(10, 11) . Unlike the
case, however, no contacts are made between the 434 repressor and the
central 6 base pairs of the 434 operator(5, 12) . We
have shown that the central base pairs in the 434 operators influence
the affinities of operators for 434 repressor by altering the
structural deformability of the DNA binding
sites(6, 11) . Our previous work in the showed that
the differing sequences in the noncontacted regions of O
1
and O
3 alters 434 repressor's ability to
discrimination between different bases at a contacted position of these
operators, presumably by modifying the structure of the operator in the
repressor-operator complexes(13) . No similar effects of
sequence-dependent differences in DNA structure on sequence recognition
by proteins have yet been reported.
Figure 1:
434 operator sequences. Sequence of
O1 and O
3 and the respective position 4 mutants
used in this study. The base pair at position 4 in the mutants is circled. Throughout this paper, operators are named by their
wild-type counterpart with the base at positions 4, 5, and/or 6
indicated where appropriate.
Analysis of the crystal
structure of R1-69 bound to O1 indicates that
glutamine 33 of repressor makes a hydrogen bond to the O-4 of the
thymine at position 4` and a van der Waals' contact with
the methyl group on the same base(5, 6) . As a result
of the base sequence difference at position 4 in O
3,
repressor can not make either contact to that base when bound to
O
3 ((6) , see Fig. 1). Since this A
T
G
C substitution at position 4 is the only sequence
difference within the contacted base pairs of O
1 and
O
3, the loss of contacts at position 4 is thought to be the
primary determinant of the 6-8-fold lower affinity of repressor
for O
3(6, 10, 13, 14) .
In agreement with this, we showed that swapping the base pair at
position 4 between O
1 and O
3 reverses their
order of affinity for repressor(13) . Our earlier results show,
however, that the energetic cost of an A
T
G
C change
at position 4 is greater in O
1 than
O
3(13) . Thus, the presence or absence of the
contact between repressor and position 4` is not the only determinant
of the difference in repressor's affinities for O
1
and O
3. Biochemical characterization of the complexes
formed between repressor and these operators revealed that the ionic
contribution to the affinity of repressor for these operators is not
solely determined by the identity of the base pair at position 4.
Instead, specific position 4/central sequence combinations direct the
ionic strength dependence and affinity order. Together these results
indicated that the loss of a contact, due to base pair substitution at
position 4, is only partly responsible for the affinity difference
between O
1 and O
3. Indeed, these
results suggest that sequence dependent variation in the strength or
number of ionic contacts between protein and DNA phosphate backbone may
have a role in modulating specific base recognition by 434 repressor.
Considering the sequences of O1 and O
3 in
light of these data reveals that the degree to which an A
T
G
C change at position 4 affects the affinity of repressor must be
modulated by the identity of the base pairs at positions 5 and/or 6
(see Fig. 1). In this paper we report the results of experiments
that directly test the role of the base pairs at positions 4-6 in
determining the degree to which 434 repressor discriminates between
O
1 and O
3. The change in operator affinity for
repressor in response to a particular mutation at position 4 depends
critically on the identity of the noncontacted base pairs at positions
5 and 6. The identity of each of these base pairs has a dramatic
influence on the contribution of ionic interactions to the affinity of
repressor for these operators. The results of other experiments
demonstrate that a contact between the amino acid at position 33 of
repressor and a 5-methyl group on a pyrimidine at operator position 4`
is critical to the affinity of operator for repressor and plays a
pivotal role in determining the ionic strength sensitivity of
repressor's affinity for position 4 variants of O
1.
Complexes between operator and Ala-mutant repressor are
unstable to filtration. Thus, in all experiments involving
Ala
, the dissociation constants were determined by
quantitative nuclease protection assays analogous to those described by (19) , but using Cu(I)-phenanthroline as the nuclease.
Cu(I)-phenanthroline protection assays were performed essentially as
described by (20) . Briefly, 3`-end labeled operator-containing
fragments were generated by cleavage of operator-containing plasmids
with HindIII and HaeIII and repair of the recessed HindIII ends in the presence of
[
-
P]dATP. This protocol generates a labeled
260-base pair operator-containing fragment and a 14-base pair labeled
fragment that does not interfere with cleavage analysis. To measure
ionic strength dependence, these DNA fragments were incubated with
varying amounts of mutant repressor in buffer containing <0.5 nM DNA, 10 mM Tris
HCl (pH 7.4), 100 µg
ml
bovine serum albumin, and 50 or 100 mM KCl. After a 10-min incubation at 23 °C, sufficient
Cu(I)-phenanthroline was added to give, on average, one cleavage/DNA
molecule in 4 min of further incubation. Following incubation with the
nuclease, the DNAs were extracted with phenol/chloroform (50:50 (v/v)),
precipitated with ethanol, suspended in 90% formamide dye mix, and
denatured by heating at 90 °C. The samples were electrophoresed on
25
30 cm, denaturing, 7.5% polyacrylamide gels containing 7.5 M urea, 89 mM Tris borate (pH 8.9) and 1 mM EDTA. Autoradiography was performed by exposing the gel to
preflashed Kodak XAR-5 film with an intensifying screen at -80
°C. Band intensities of the footprinting results were quantified,
and the dissociation constants were determined from at least four
different autoradiograms generated from separate experiments.
Repressor-O1-4G
C complexes are
not stable to either filtration or Cu(I)-phenanthroline digestion,
hence quantitative DNase I footprinting was used to determine the
affinity of 434 repressor for
O
1-4G
C, essentially as
described(19) . To generate the
O
1-4G
C operator, a 30-base
oligonucleotide of the sequence
5`-GGGGTCGAACAAGAAAGT
CTGTTCGACCCC-3` and its complement
were subjected to electrophoresis through a denaturing 20%
polyacrylamide gel and purified on DEAE paper according to the
manufacturer's instructions (Schleicher & Schuell). One
strand was then 5`-end labeled with [
-
P]ATP
and polynucleotide kinase, annealed to its complement, and the
resulting double-stranded product was purified on a nondenaturing 20%
polyacrylamide gel. Following further purification on DEAE paper, this
30-base pair operator-containing duplex was used in DNase I
footprinting experiments. Binding reactions were carried out in a
buffer identical to that used in the filter binding experiments but
supplemented with 0.1 mM MgCl
(Mg
enhances DNase I activity). To control for any effects of DNA
length and the presence of Mg
in the binding buffer,
identical binding reactions were carried out on control DNAs generated
by cleavage of plasmids carrying O
1-4A and
O
1-4G with HindIII and SmaI
followed by gel purification of the resulting 51-base pair
operator-containing fragment and repair of the recessed HindIII end with the Klenow fragment of DNA polymerase I in
the presence of [
-
P]dATP. After a 10-min
incubation of these DNAs with varying amounts of repressor at 23
°C, sufficient DNase I was added to give, on average, one
cleavage/DNA molecule in 5 min of further incubation. Following
incubation with the nuclease, the DNAs were extracted with
phenol/chloroform, precipitated with ethanol, suspended in 90%
formamide dye mix, and denatured by heating at 90 °C. The samples
were then treated as described above. The dissociation constants were
determined from three different autoradiograms resulting from separate
experiments.
We showed previously that ionic interactions play a much
greater role in stabilizing the complexes formed between repressor and
the position 4 variants of O1 and O
3 than they
do in complexes formed between repressor and the wild-type operators.
To determine the extent to which the identity of the base pair at
position 6 affects the ionic interactions of repressor with
O
1, O
3 and their position 4 variants, the
dependence of K
on monovalent cation concentration
was measured. In O
1, when the base at position 4 is an
A
T, a position 6 C
G
T
A change results in an
operator (O
1-4A6T) whose affinity for repressor
decreases 11-fold when the ionic strength is shifted from 50 to 100
mM KCl. This contrasts with the insensitivity of the affinity
of repressor for wild-type O
1 (O
1-4A6C)
to changes in ionic strength (Table 1). Conversely, when position
4 is occupied by a G
C base pair in O
1, the position 6
C
G
T
A change results in an operator
(O
1-4G6T) whose affinity for repressor is only
moderately decreased (2.5-fold) by increasing the ionic strength from
50 to 100 mM KCl, while under identical conditions the
affinity of repressor for O
1-4G6C decreases by
18.5-fold. Similar sequence-dependent effects are observed for the
position 4 and 6 variants of O
3. When the base pair at
position 4 of O
3 is a G
C, changing the base pair at
position 6 from a T
A to a C
G increases the ionic strength
dependence of the repressor-operator complex from one that is
essentially independent of salt concentration to one that is
destabilized 11-fold as the salt concentration increases from 50 to 100
mM KCl. As seen in the context of O
1, changing
position 6 has the opposite effect on ionic strength dependence of
affinity when position 4 is first mutated; when position 4 in
O
3 is an A
T, changing the base pair at position 6
from a T
A to a C
G decreases the ionic strength sensitivity
of repressor's affinity. Together, the data from the above
analyses show that neither the base pair at position 4 nor the base
pair at position 6 specifies the ionic strength dependence of the
affinity of repressor for these operators. Instead, a specific
combination of position 4 and position 6 base pairs determines the
ionic strength dependence of the affinity of repressor.
The
differing ionic strength dependences of complexes formed between
repressor and the position 6 mutants of the wild-type and position 4
variant O1 and O
3 indicate that the position 6
base pair has a major role in determining the degree that ionic
interactions contribute to operator strength. Differences in the ionic
strength dependences of a protein-DNA complex can be due to changes in
the strength and/or number of ionic interactions between protein and
DNA. Since all 434 repressor-operator complexes studied thus far appear
to have an identical number of contacts with the DNA phosphate backbone (5, 6, 21, 22, 23) , the
observed differences in ionic contributions to binding energy must be
due to an alteration in the strength of ionic interactions. This
observation implies that the structures of these repressor-operator
complexes differ and that these structural differences are mediated, at
least in part, by cooperative interaction between positions 4 and 6
base sequences.
Fig. 2presents data comparing
the affinity of repressor at 50 and 100 mM KCl for operators
with either GC, A
T, T
A, or C
G base pairs at
position 6 in the context of either an A
T or a G
C base pair
at position 4 in both O
1 and O
3. In the context
of an A
T base pair at position 4 (Fig. 2, A and C) a T
A at position 6 is not the only base pair that
results in an operator whose affinity for repressor is significantly
dependent upon ionic strength. In the context of a position 4 A
T,
only a position 6 C
G markedly reduces the ionic strength
dependence of the affinity of repressor for these operators. Likewise,
in operators with a G
C base pair at position 4 (Fig. 2, B and D), a position 6 T
A is not the only base
pair that results in an operator whose affinity for repressor is only
weakly dependent upon ionic strength. In O
1-4G, both
a G
C and a T
A base pair permit repressor to bind in a
fashion that is relatively independent of ionic strength. Similarly in
O
3-4G, only a position 6 C
G base pair generates
an operator whose affinity for repressor is markedly dependent upon
ionic strength. Consideration of the data in Fig. 2also shows
that a position 6 T
A is not the only base pair that can suppress
the deleterious effect of an A
T
G
C change at
position 4 on the affinity of repressor for operator. In the context of
O
3, at both 50 and 100 mM KCl, changing position 6
from a T
A to a G
C or an A
T also results in an
operator whose affinity for repressor is relatively unaffected by an
A
T
G
C change at position 4 (compare Fig. 2, C and D). In this sequence context, only a position 6
C
G base pair results in an operator whose affinity for repressor
is strongly decreased by position 4 substitution. Comparing Fig. 2, A and B, reveals a qualitatively
similar but not quantitatively identical pattern of position 6 effects
on discrimination at position 4 in O
1. Taken together,
these data do not support a model where the participation of the base
pair at position 6 in an oligo-dA
oligo-dT tract defines its role
in determining either the affinity of repressor or the ionic strength
sensitivity of the complexes between repressor and position 4 variants
of O
1 and O
3.
Figure 2:
Effect of position 6 substitution on the
affinity of 434 repressor for O1, O
3 and their
position 4 variants. Plotted is the K
versus the base at position 6 in O
1 (A), O
1-4G (B),
O
3-4A (C), and O
3 (D).
Values of the dissociation constants measured at 50 (hatched
bars) and 100 (filled bars) mM KCl are shown.
Note scale difference in panelsA and C from B and D
In O1,
position 5 is occupied by an A
T base pair, and in O
3
position 5 is a T
A. At 100 mM KCl, changing the base
pair at position 5 of wild-type O
1 from an A
T to a
T
A decreases the affinity of repressor for the resulting
operator by
2-fold (Table 2, compare lines 1 and 5).
Conversely, in O
3, the same position 5 A
T
T
A change increases the affinity of repressor for the
resulting operator by
4-fold (Table 2, compare lines 12 and
16). Thus, the net result of swapping the base pairs at position 5
between wild-type O
1 and wild-type O
3 is to
increase the degree to which repressor discriminates between these
operators by
2-fold.
By comparing the ionic strength dependence
of the affinity of repressor for two operators with the same position 4
and position 6 base, we can deduce the degree to which a TA versus an A
T base pair at position 5 contributes to the
affinity of repressor for each operator and the ionic strength
dependence of repressor-operator complexes. Similarly by comparing the
effect of an A
T
G
C change at position 4 in the
context of identical base pairs at position 6, we can determine the
contribution of the base pair at position 5 to position 4 preference.
Sequential comparisons of this type reveal the context-dependent nature
of the effect of a base pair substitution at position 5.
When the
base pair at position 4 is an AT, in every position 6 context
except one (6T), the presence of a T
A base pair at position 5
results in a binding site whose affinity for repressor is more ionic strength dependent than when position 5 is an A
T (Table 2, compare lines 1-4 with 5-8). Conversely,
when the base pair at position 4 is a G
C, in every position 6
context, a position 5 A
T
T
A substitution results in
an operator whose affinity for repressor is less dependent on
ionic strength (Table 2, compare lines 9-12 with
13-16). Furthermore, in the context of a position 4 A
T, a
position 5 A
T
T
A substitution consistently alters
the ionic strength dependence of the affinity of repressor for operator
by only
1.5-fold (Table 2, compare lines 1-4 with
5-8). The same substitution lowers the ionic strength dependence
of the affinity of repressor for operators with a position 4 G
C
by as little as 1.5-fold (Table 2, compare line 9 with 13) to as
much as 45-fold (Table 2, compare line 11 with 15). Taken
together, these results demonstrate that the degree to which the
affinity of repressor for position 4 variants is altered by ionic
strength is completely dependent upon the identity of the base pairs at
positions 5 and 6. Moreover, the effects of the base pairs at positions
4-6 are not independent of each other; specific combinations of
base pairs at these three positions cooperate to generate different
ionic strength dependence phenotypes.
Although the magnitude of the
effect of a position 4 AT
G
C change differs
depending on the base pair at position 6 (for example see Table 1), in every position 6 context the identity of the base
pair at position 5 only slightly influences the magnitude of the
affinity change resulting from this mutation at position 4. For
example, at high salt, when the base pair at position 6 is a C
G,
changing the base pair at position 4 from an A
T to a G
C
lowers repressors affinity by 175-fold in the context of an T
A
base pair at position 5 (Table 2, compare lines 5 and 13).
Similarly, the same position 4 change has a 195-fold effect on affinity
in the context of a position 5 A
T base pair (Table 2,
compare lines 1 and 9). While changing position 5 does not greatly
affect the magnitude of repressor's position 4 preference,
changes at position 5 do influence repressor's position 6
preference. For example, in an operator bearing a position 4 G
C,
at high salt, repressor prefers a T
A base pair at position 6 over
an A
T by
10-fold in the context of an A
T at position 5 (Table 2, compare lines 11 and 12), while when position 5 is
occupied by a T
A base pair, repressor now shows a 3-fold
preference for an A
T over a T
A base pair at position 6
(lines 15 and 16). Thus, mutating position 5 can change
repressor's position 6 preference by 30-fold. A 15-fold change in
position 6 preference is mediated by altering position 5 is the context
of a position 4 G
C ( Table 2compare lines 10 and 11 with 14
and 15). In addition to these position 5-dependent reversals in the
affinity order of repressor for position 6, swapping a T
A for an
A
T at position 5 can also produce more subtle effects on the
magnitude of repressor's position 6 preference; for example,
compare 4G5T6CT with 4G5A6CT ( Table 2compare lines 13 and 16
with 9 and 12). Taken together, these data show that position 5 has a
much greater effect on repressor's discrimination at position 6
than it does on discrimination at position 4.
Previous work has shown that 434 repressor distinguishes
between O1 and O
3 in large measure because of
the sequence difference at position 4 of these
operators(8, 13, 14) . The results presented
here show that the affinity of repressor for O
1 and
O
3 and two of their position 4 variants is strongly
influenced by the base pairs at positions 5 and 6. Moreover, the degree
to which position 4 substitutions affect operator strength is tempered
by the identity of the base pairs at positions 5 and 6. We have shown
here that these base pairs influence the affinity of repressor by
modulating the degree to which ionic interactions contribute to the
stability of the repressor-operator complex.
The influence of the
base pair at position 6 on the affinity of repressor for an operator is
exemplified by the observed effects of base pair substitutions at that
position on the affinity of repressor at high salt concentration. The
base pair at position 6 that results in lower or higher
repressor-operator affinity depends upon which base pair is at position
4 (Table 1). Moreover, though 434 repressor discriminates between
wild-type O1 and O
3 by
5-fold throughout
the salt concentration range studied ( Table 1and (13) ),
our results show that a position 6 swap between O
1 and
O
3 generates operators whose affinity for repressor now
differs by
45-fold in this range of salt concentration.
Furthermore, if O
1 and O
3 were to have both a
C
G base pair at position 6, then repressor would discriminate
between them by
40-fold at 50 mM KCl and 350-fold at 100
mM KCl. These data clearly demonstrate that the identity of
the base pair at position 6 of O
1 and O
3 plays
an important role in determining the degree to which repressor
discriminates between these operators. The identity of the base pair at
position 5 of O
1 and O
3 plays a similar, though
less dramatic, role in determining the degree to which repressor
discriminates between these operators. The net result is that swapping
the base pairs at position 5 between wild-type O
1 and
wild-type O
3 increases the degree to which repressor
discriminates between these operators by
2-fold (Table 2).
Changes at position 5 have a much larger effect on repressor's
position 6 preference. Moreover, since the base pair at position 6
alters or even reverses repressor's position 4 preference and the
base pair at position 5 modulates position 6 preference, position 5
indirectly influences repressor's position 4 preference. This
influence is only apparent in certain position 6 contexts. Therefore,
the base pairs at positions 5 and 6 combine to influence
repressor's preference at position 4.
Since neither the major
nor minor groove surface of the base pairs at positions 5 and 6 in
O1 and O
3 are contacted by
repressor(6, 10) , we suggest that the base pairs at
positions 5 and 6 modulate repressor's affinity for operator by
changing operator structure in the complex. Dissimilar salt dependences
are thought to be the result of differences in the number and/or
strength of charged interactions between protein and the DNA phosphate
backbone(28) . Two lines of evidence support the idea that
differences in the ionic strength sensitivity of repressor's
affinity for position 4 variants of O
1 and O
3
result from alterations in the strength of a shared number of ionic
contacts rather than a difference in the actual number of contacts. The
number of protein-phosphate backbone contacts identified by
ethylnitrosourea interference experiments is identical for all
repressor complexes tested(21, 22) . In addition,
comparison of the crystal structures of amino acids 1-69 in
complex with O
1, O
3, and
O
1-4G reveals an identical number of direct
interactions between the protein and the phosphate backbone of these
operators(5, 6) . (
)Assuming that all
repressor-operator complexes studied here have the same number of ionic
interactions, an increase in the ionic strength sensitivity of a
particular repressor-operator complex, as a result of changes in
operator sequence, indicates that the change in operator sequence
caused a reduction in the strength of ionic interactions within that
complex. In this view, the base pairs at positions 5 and 6 modulate the
affinity of repressor for position 4 variants of O
1 and
O
3 by altering the contribution of ionic interactions to
the overall binding energy. Our data also show that the combination of
base pairs at positions 5 and 6 that result in a lower or higher
dependence of repressor-operator affinity on ionic strength depends
upon which base pair is at position 4.
The simplest interpretation
of these results, in light of this structural model, is that the
sequence-dependent structural alterations conferred by a particular
combination of bases at positions 5 and 6 that generate a favorable
configuration of the phosphates when position 4 is an AT must
result in a less favorable configuration when position 4 is a G
C.
In agreement with this, differences in the sensitivity of a
repressor-operator complex to cleavage by hydroxyl radical depend upon
the position 5 and 6 context of the position 4 base (13) . We
show here that these dissimilar ionic strength sensitivities are a
result of the base pair composition of O
1 and
O
3 at positions 5 and 6.
An alternative explanation of
the ionic strength and affinity data suggests that instead of
reflecting changes the protein-DNA interface, the effect of position 4,
5, and 6 changes on the strength of repressor-operator complexes may
indicate alterations in the conformation of repressor's dimer
interface. Support for this idea comes from the observation that
mutagenizing amino acids involved in forming the amino-terminal dimer
interface can alter repressor's central sequence
preferences(11) . The data presented here can
neither prove nor dismiss this alternative. However, the observed
differences in ionic strength sensitivity arise from base pair changes
near the locations of known protein-DNA phosphate backbone interactions (10) . Moreover, these differences in ionic strength
sensitivity are correlated with changes in the structure of the DNA
within these operators(13) . These observations suggest that
changes in operator sequence at positions 4-6 alter the
interactions between the repressor and the operator DNA phosphate
backbone.
The observation that a Gln
Ala
mutation allows repressor to form complexes with
O
1-4G and O
3-4A that are relatively
insensitive to salt concentration implies that the strong ionic
strength dependence of O
1-4G- and
O
3-4A-wild-type repressor complexes is due to altered
contacts between glutamine 33 and the position 4 base. The precise
nature of this structural alteration can not be determined from our
data. Our data do suggest, however, that simply losing a van
der Waals' contact with the base at position 4` is not sufficient
to cause the stability of a repressor-operator complex to become
sensitive to ionic strength. For example, the affinities of repressor
for wild-type O
3 or O
1-4G-6T are not
greatly affected by changes in salt concentration, even though no van der Waals' contact between Gln
and the
base at position 4` appears possible. Why then are some of wild-type
repressor-operator complexes affected by ionic strength, but none of
the Ala
-repressor-operator complexes are? We suggest that
Ala
may make some residual contact with position 4 in all
operators, and it is this contact that prevents ionic strength from
affecting complex formation. Support for the suggestion that Ala
contacts the base at position 4 comes from measurements of the
affinity of Ala
for a set of symmetric reference operators
in which the base pair at position 4 of each half-site (positions 4 and
11` in Fig. 1) is varied(8) . These results demonstrate
that Ala
is capable of strongly distinguishing between
A
T, A
U, G
C, and G
C base pairs at
position 4 of these operators. This observation implies that, in some
way, the base pair at position 4 is recognized by the Ala
protein.
The idea that some form(s) of contact between the position 4 base and amino acid 33 can reduce the ionic strength sensitivity of a repressor-operator complex suggests a possible explanation for the observed effects of position 5/6 base pair substitutions. That is, only particular position 5/6 base pair combinations permit repressor's amino acid 33 to make a contact with the position 4 base pair, thereby rendering the stability of the complex relatively impervious to changes in salt concentration, while other position 5/6 combinations do not. Alternatively, the status of the contact at position 4 could be solely determined by the identity of the base pair at that position, while the overall affinity and ionic strength sensitivity of the resulting complex is modulated by the response of the repressor to the position 5/6 sequence context in which that contact is (or is not) made. The results presented here neither support nor exclude either mechanism.
The idea that the contact at
position 4 has a role in establishing the ionic strength sensitivity of
repressor's affinity is directly supported by the observation
that replacing the position 4` C in O1-4G with a
C both increases operator affinity for repressor and
decreases the ionic strength dependence of the affinity of repressor
for O
1-4G. The independence of repressor's
affinity for O
1-4G
C from salt
concentration demonstrates that reestablishment of a contact involving
the 5-methyl group at position 4` is sufficient to eliminate the strong
ionic strength sensitivity observed for the affinity of repressor for
unmodified O
1-4G (Table 4). Furthermore, the
fact that the ionic strength sensitivities of the
repressor-O
1-4A
T and
repressor-O
1-4G
C complexes are
indistinguishable suggests that the presence of a 5-methyl group at
operator position 4` is sufficient to generate ionic strength
insensitivity in the context of an O
1 central sequence.
Taken together, our results imply that all of the bases that differ
between O1 and O
3, positions 4, 5 and 6, are
important in determining repressor's affinity. A difference in
base sequence at position 4 is relevant because it determines, at
least, the presence or absence of a direct contact with repressor.
Positions 5 and 6 are relevant because their identity modulates the
ionic contribution to the binding energy. Moreover, there is an
intimate connection between the status of the contact at position 4 and
the nature of the influence of the base pairs at positions 5 and 6 on
the affinity of repressor. The sequence of the noncontacted
bases at operator positions 5 and 6 can modify preference of 434
repressor for a base at a contacted position. As suggested by the ionic
strength dependence data shown here and our earlier
results(15) , the effect of these noncontacted bases is
presumably through alteration of the DNA structure in the
repressor-operator complex.
Having established that positions 5 and
6 modulate repressor's position 4 preference in a manner that
depends on ionic strength leads to the question of why the phage
evolved the particular combination of bases found at positions
4-6 of O1 and O
3. One possible answer to
this question emerges from consideration of our data in light of recent
measurements which establish that E. coli's cytoplasm
contains
140 mM K
(29) . Our
results suggest that at this salt concentration, changes in the base
sequence at positions 4, 5, or 6 of O
1 or O
3
would result in a greater separation of repressor's affinity for
these operators. Given that the genetic switch governing 434 phage
development requires maintaining a small difference in the affinity of
repressor for O
1 and
O
3(1, 2) , it is striking that by making
use of sequence-dependent modulations of both ionic and nonionic
interactions in the repressor-operator complex the phage has evolved a
pair of operators that fulfill this requirement.