(Received for publication, July 19, 1995; and in revised form, October 2, 1995)
From the
Bacteriophage promoters P
and P
direct RNA synthesis in divergent orientations from start sites
82 base pairs apart. We had previously determined that the presence on
the same DNA fragment of a wild type P
promoter interfered
with the utilization of the P
promoter. The results
reported here concern the effects of changing the distance between the
start sites by insertion or deletion of 5 or 10 base pairs. Three
different techniques (run-off transcription, gel mobility shift, and
permanganate probing) were employed to monitor complex formation at
P
. Unexpectedly we find that deletion of 10 base pairs
between the start sites abolishes the interference, whereas insertion
of 10 base pairs does not. Deletion of 5 base pairs, however,
essentially prevents joint complex formation at P
and
P
. These findings suggest several ways in which for the
wild type separation of the two promoters the utilization of P
could be affected by an RNA polymerase at P
. In
addition to direct steric interference, these include the obstruction
of access to DNA sites necessary for optimal contact with the RNA
polymerase.
Promoters P and P
of bacteriophage
direct the synthesis of nonoverlapping, divergent transcripts
originating from start sites separated by 82 base pairs. We (1, 2) as well as Gussin and co-workers (3, 4) have shown that the presence of the P
promoter has a negative effect on the ability of RNA polymerase
to form open complexes at P
. The use of P
mutants allowed Gussin's group to demonstrate that
conversely, the presence of an RNA polymerase at P
negatively affects the utilization of a (weakened) P
promoter as well (5) .
Open complex formation at the
wild type P promoter occurs within
seconds(6, 7) , but formation of the complex at
P
is several orders of magnitude slower, on the time scale
of tens of minutes(1, 3, 8) . Thus nearly
every RNA polymerase binding at P
does so in the context
of another polymerase situated at P
. Our own work (1, 2) as well as more extensive kinetic analysis by
Gussin and his group (3, 4) has demonstrated that the
interference with complex formation at P
manifests itself
not at the initial bimolecular (binding) step but rather in some
subsequent step involving conformational transitions in RNA polymerase
and/or DNA. The interference slows down but does not prevent open
complex formation at P
; ultimately open complexes at both
promoters do co-exist on the same DNA fragment ( (1) and (9) and this work).
Reduction of the distance between
P and P
by one base pair further slows open
complex formation at P
(4) . The effects of
considerably shorter separation between the P
and P
promoters can be assessed from studies on other lambdoid phages.
For the 434 phage where the distance between the start sites of P
and P
is about 65 base pairs, the -35 regions
of the two promoters overlap and concurrent binding of RNA polymerases
at the two promoters is not observed(10) . For P22 the
interpromoter distance is even shorter, making it quite unlikely that
concurrent binding of two polymerases could occur(11) . To
establish how the P
promoter affects P
function over a range of distances not covered in the above
studies, we have investigated open complex formation at P
using constructs with 5- and 10-base pair insertions or deletions
between the -35 regions of the phage
P
and
P
promoters. Surprisingly, we found that the variant with
the 10-base pair deletion in the region between the two promoters,
leaving just two base pairs between their -35 regions, was
essentially impervious to the presence or the absence of a functional
P
promoter on the same DNA fragment.
Figure 3:
Analysis of complex formation by gel
shift. A, autoradiograph of a gel demonstrating the
time-dependent formation of two complex bands. DNA fragment (1
nM) and 200 nM RNA polymerase were incubated at 37
°C for the indicated amount of time, after which the binding
reaction was terminated by the addition of heparin. The first complex
band (assigned as open complex formation at P and labeled
as the 1RPo complex) is practically instantaneous; appearance
of the 2RPo band (with two bound RNA polymerases) reflects subsequent
open complex formation at P
. B, analysis of
kinetic data obtained from gels such as those shown above. The
intensity of the 2RPo complex was taken as a measure of P
occupancy. The symbols reflect experimentally determined
values; the curves drawn represent best fits to the data, as
follows: open circles, Wt, k
=
0.03 min
; filled circles, Wt
P
, k
= 0.09
min
; open triangles, D10, k
= 0.24 min
; filled triangles, D10 P
, k
= 0.10
min
.
Figure 1:
The sequences of the variants used in
this work. The construct designated Wt has the Wt P and P
promoters, with an 82-base pair separation
between the start sites. The -10 and -35 regions of
P
are boxed as shown. The -35 regions of
P
are doubly underlined, and the -10 regions
are singly underlined. The designation
P
refers to DNAs that contain an
inactivating T
C substitution at position -7 (the
``constant T'') of P
. Insertions and deletions
have been introduced, as shown, into the DNA separating the -35
regions of P
and
P
.
DNaseI
footprinting of species with particular electrophoretic mobilities was
carried out by pretreatment of complexes with the nuclease after the
addition of the heparin, in transcription buffer containing 10 mM MgCl (instead of 3 mM) to increase the
cutting efficiency of the DNase. Upon electrophoretic separation of the
nuclease treated complexes, the DNA was isolated from individual bands
and analyzed on sequencing gels as described(1) . In some
experiments the binding of RNA polymerase to an unidentified DNA region
resulted in the appearance of an extra complex band in mobility shift
assays. The relatively fast formation of this complex necessitated the
footprinting of the equivalent of a 3RPo complex in lane 3 (but no other lanes) of Fig. 4. As the results are
identical to our other results on 2RPo complexes(1) , the
presence of the uncharacterized extra RNA polymerase apparently does
not affect the results in any way.
Figure 4:
DNaseI footprinting pattern of the DNA in
the complex bands as detected in gel shift assays. The approximate
positions of the P and P
promoter have been
indicated on the figure. The DNA in various bands on the gels was
extracted and loaded on a 6% polyacrylamide/6 M urea gel for
analysis of the DNaseI cutting patterns. Lanes 1-3, Wt
DNA; lanes 4-6, D10 DNA; lanes 7-9, I10
DNA. Lanes 1, 4, and 7, free DNA; lanes
2, 5, and 8, the 1RPo band; lanes 6 and 9, the 2RPo band; lane 3, a 3RPo band with one RNA
polymerase at an unknown site (see ``Experimental
Procedures'').
The DNA sequences between the start sites of P and P
are shown in Fig. 1for the constructs
used in this work, aligned with respect to the start site at the
P
promoter. The actual ligated fragments cloned into
pKK232-8 extended some 6 base pairs beyond the start site of each
promoter. In addition to the wild type spacing between P
and P
, we constructed templates with deletions (D5
and D10) or insertions (I5 and I10) between the -35 regions of
P
and P
. Assuming the DNA is B-form with 10.5
base pairs/turn, for the Wt, D10, and I10 constructs, the centers of
the -35 regions of P
and P
are
under-rotated by about 100 ° with respect to each other, but for D5
and I5 they are over-rotated by about 80 °. Thus the -35
regions are separated by similar angles in both cases but approach each
other from opposite sides. To address the effect of RNA polymerase
binding at P
on open complex formation at P
for the fragment with wild type, I10, and D10 spacings, we also
made variants for which P
had been inactivated by a point
mutation in the -10 region. All constructs are identical to each
other with respect to the P
promoter from the -35
region and downstream.
To determine the extent to which the P and P
promoters are utilized on each of the DNA
constructs, single round run-off transcription reactions were carried
out using the wild type template and those with the 10-base pair
insertion or deletion (I10 and D10, respectively). The experimental
protocol involves the addition of heparin (in order to inactivate free
RNA polymerase and that in closed promoter complexes) 5 or 60 min after
mixing the DNA and RNA polymerase, followed by the addition of the
nucleoside triphosphates. Thus the RNA synthesized would be derived
from open complexes formed in the time interval ending with the
addition of heparin and be a measure of the extent to which open
complexes had formed during the interval. The results are shown in Fig. 2. The sizes of the run-off products from P
and P
are not affected by the context of the
promoter, indicating that the same start sites are used on the variants
D10 and I10 (and I5; no RNA product was obtained with D5 (data not
shown)) as with the wild type DNA sequence. As expected from previous
work, inactivation of the P
promoter (evident from the
disappearance of the corresponding bands in Fig. 2(lanes marked P
)) facilitated
utilization of the P
promoter on the fragment with wild
type (82-base pair) spacing between P
and P
(Fig. 2, compare lanes marked Wt).
Surprisingly however, the intensity of the P
-derived band
was greatly enhanced on D10 as compared with the wild type or I10
constructs. Inactivation of the P
promoter served as an
equalizer, as is evident from the last six columns of Fig. 2;
the mutation lead to greatly enhanced intensities of the P
bands on the wild type and I10 templates to levels more akin to
those on D10 with or without the P
mutation.
Figure 2:
Single round run-off transcription
reactions show a favorable effect of a 10-base pair deletion between
P and P
on utilization of P
. The
positions of the P
and P
transcripts (the
latter a doublet in line with previous experiments) are indicated. The
times of 5 and 60 min refer to the duration of the incubation of DNA
and RNA polymerase prior to initiation of RNA synthesis. In the lanes labeled P
, note
the appearance of a larger transcript, which likely originated at the
P
promoter described by Gussin and
co-workers(22) .
Even though
the interpromoter distance on the D10 fragment is 10 base pairs shorter
than that on the wild type one, no interference from the P promoter on P
function was observed in the above
experiments. In view of the fact that P
and P
could concurrently be occupied for the wild type separation
between them(1, 9) , it was of interest to determine
whether on the D10 template this would be the case as well. We
addressed this question using gel shift analysis (Fig. 3A) and found that the two more slowly moving
complexes that could be detected for both the wild type and the D10
DNAs had similar mobilities. Previously we had established that the
slowest moving complex contained RNA polymerases at both P
and P
(1) . We therefore interpret these
results as indicative that on the D10 construct, joint occupancy of the
two promoters occurs as well. This was confirmed by DNaseI footprinting
of the more slowly moving complexes for the Wt, D10, and I10 fragments
(see Fig. 4). The footprint pattern determined for the Wt DNA is
similar to that previously reported for this DNA(1) ,
demonstrating the concurrent protection of the P
and the
P
promoters. Such a pattern is also observed for the
two-polymerase complexes at D10 and I10, providing further evidence of
their ability to concurrently bind a polymerase at each promoter. In
addition some subtle differences in the footprints of complexes on the
three templates are seen that may be related to template-dependent
differences in the extents to which the regions covered by RNA
polymerase at the two promoters overlap. With complexes protected at
just P
, two similarly positioned bands of enhanced nuclease
sensitivity are observed for all three DNAs, whereas I10 has an
additional third hypersensitive site further upstream from P
(compare lanes 2, 5, and 8 of Fig. 4). With complexes that show protection at P
as well, differences among the three templates are observed in
the extent to which the hypersensitivity of the bands is suppressed
(see lanes 3, 6, and 9 of Fig. 4).
The gel mobility shift technique also enabled us to follow the
kinetics of open complex formation at the P promoter.
Visual inspection of the data in Fig. 3A suggests that
formation of two-polymerase complexes (i.e. RNA polymerase
binding concurrently at P
and P
) is much
faster on the D10 template than on the one with wild type spacing. As
in both cases complex formation at P
is instantaneous on
the time scale of these experiments; this difference reflects the
different rates of complex formation at P
. Quantitative
analysis of the results is presented in Fig. 3B, which
clearly shows the much faster rate of complex formation at P
on the D10 template as compared with the wild type template.
Conversely, the two templates show very similar curves for complex
formation at P
in the context of an inactivated P
promoter. The rate constants determined by these and other gel
mobility shift experiments are presented in Table 1(first
column).
In view of the surprising result that shortening the
interpromoter distance between P and P
can
lead to an enhanced rate of complex formation at the P
promoter, we have investigated the strand separation process by a
direct technique, that of probing with permanganate(15) . T
residues in single stranded DNA are much more sensitive to the
oxidizing agent than those in double helical DNA. Thus the RNA
polymerase-induced melting of the DNA spanning the start site and the
-10 element can be detected by the increase in sensitivity of
these residues. In Fig. 5the patterns of reactivity are
displayed for all eight promoter variants shown in Fig. 1after
incubation of each DNA with RNA polymerase for 5 or 60 min. The almost
complete inactivation of the P
promoter by the single base
substitution at position -7 is again readily apparent; only very
weak bands indicative of RNA polymerase-induced enhanced cutting can be
seen in the lanes containing this mutated DNA. It is clear that RNA
polymerase leads to similar patterns of enhanced DNA breakage at
P
in the context of the five different spacings
investigated, regardless of whether P
is inactivated or
not. From sequencing reactions using the same primer as was used here
to label the bottom strands (Fig. 1) in polymerase chain
reaction reactions, (
)we assigned the four most prominent
bands in the P
(lower) region of the gel from top to
bottom to T residues at positions -11, -9, -5, and
-4, respectively (see Fig. 5). The similarity of the
patterns indicates that the region of strand separation must be similar
for all the constructs and that RNA polymerase must be positioned
similarly on the P
promoters of each. The data displayed
in Fig. 5also show that ``half-turn'' insertion or
deletion as in I5 and D5 have entirely different effects. With I5, open
complex formation at P
is clearly detectable, but the
deletion of 5 base pairs (as in D5) essentially renders open complex
formation at P
unable to be achieved, presumably due to
interference from the RNA polymerase bound to P
.
Figure 5:
Determination of strand separation at
P and P
by permanganate probing. The various
DNAs were end-labeled in the bottom strand of Fig. 1. Complex
formation was allowed to proceed at 37 °C for 5 or 60 min prior to
addition of heparin. After exposure to permanganate (7 mM) for
2 min and cutting with piperidine, the products were separated on a
denaturing gel. The lanes contained samples as indicated. The
results were derived from two different gels; the experimental
conditions were similar for both. Assignments of bands was by
comparison with sequencing gels (not
shown).
We have
used the appearance of RNA polymerase-induced permanganate sensitivity
as another means for monitoring the kinetics of open complex formation
at P. The results for the Wt, D10, and I10 templates are
shown in Fig. 6a, those for these same templates but
with an inactivated P
promoter in Fig. 6b.
Again the faster formation of an open complex at P
on the
D10 template is readily apparent, as is the acceleration of the rates
on I10 and Wt DNA by the inactivation of P
. The rate
constants obtained by computer fitting of the results are shown in Table 1(second column). The reasonable agreement between the
values obtained here and those from the gel shift experiments indicates
that both methods must be monitoring the same process, namely formation
of open complexes.
Figure 6:
Kinetics of open complex formation at the
P promoter as determined by the permanganate assay. The
aggregate intensity (expressed as the percentage of the total amount of
radioactivity on the gel) of the four bands resulting from modification
at positions -11, -9, -5, and -4 was used as a
measure of complex formation. The curves were drawn for the
best fit values of k
as indicated below. a, the P
promoter is wild type. Open
triangles, D10, k
= 0.19
min
; open circles, Wt, k
= 0.05 min
; filled circles,
I10, k
= 0.02 min
. b, the P
promoter has been inactivated by
mutation. Open triangles, D10 P
, k
= 0.15 min
; open
circles, Wt P
, k
= 0.09 min
; filled circles,
I10 P
, k
=
0.04 min
.
We have also performed kinetic studies by the
single round run-off assay, following the rate of appearance of the
transcripts originating at P and P
as a
function of the time of incubation of the DNA with RNA polymerase prior
to termination of the reaction by the addition of heparin. As is
apparent from Table 1(third column), in general the rates
measured by the run-off assay are smaller than those observed by the
gel shift and permanganate probing techniques, and the differences
between the various templates are less pronounced. In prior studies (17) on other variants of the P
promoter, we had
observed a similar trend in comparing kinetic results from run-off
experiments with those obtained by the abortive initiation assay. These
findings suggest that in the run-off experiments a slower process not
observed in the measurements by the other two methods is rate-limiting.
We have not studied this phenomenon further, but likely candidates for
such a process are initiation of RNA synthesis and promoter clearance.
The fits to the run-off data did indicate a significant
(2-3-fold) increase in the plateau values of the kinetic curves
upon inactivation of P
, thus reconciling the similarity in k
values evident in Table 1with the large
differences in band intensities shown in Fig. 2. Finally it
should be pointed out that for the wild type template the values of
calculated from the reciprocals of the k
values presented in Table 1(a range of
25-50 min for the three techniques) are in good agreement with
values obtained by others for this promoter(3) .
We have shown by three independent assays that open complex
formation at P benefits from a 10-base pair reduction in
the distance between the P
and P
promoters.
This is a surprising result, as it had previously been demonstrated
that the presence of a functional P
promoter interfered
with P
function even for the wild type 82-base pair
separation between the start sites of the two promoters. Our results
indicate that with a DNA construct for which the separation is reduced
to 72 base pairs, open complex formation at P
is not
inhibited by the presence in cis of a functional P
promoter. Because open complex formation at the wild type P
sequence is very fast, this implies that on the D10 template the
RNA polymerase engaging in open complex formation at P
is
essentially impervious to the presence of an RNA polymerase at
P
. The open complexes formed at P
on the D10
template and also the other variants shown in Fig. 1are similar
to those formed on the wild type template with respect to both the T
residues at which base pairing is disrupted and the start site for RNA
synthesis. Thus the creation of new promoters as a consequence of the
sequence alterations cannot be invoked in explaining the observations
reported here.
The observed relief of promoter interference by a
reduction in the distance between two promoters suggests two ways that
are not mutually exclusive by which an RNA polymerase at P could affect an RNA polymerase in the process of formation of an
open complex at the P
promoter: steric interference and
occlusion of DNA sites contacted by P
-bound RNA
polymerase. In the former case, possibly the 10-base pair deletion
allows two RNA polymerases to bind at the adjacent promoters without
unfavorable contacts between them, as shown schematically in Fig. 7. In order to conform to the kinetic results reviewed in
the introduction(1, 3, 4) , no interference
would occur in the intermediate (closed) complex. There is indirect
evidence (7) for the occurrence of a conformational change in
RNA polymerase in the process of formation of an open promoter complex
after the initial contact between the enzyme and promoter DNA. This
process (shown as the straight arrows in Fig. 7) would
be slowed down at P
due to interference of the polymerase
at P
. The occurrence of steric interference is a reasonable
possibility in view of the size of RNA polymerase as deduced from the
DNaseI footprints at the promoters. The individual footprints of RNA
polymerase at P
and P
each extend at least 50
base pairs upstream of the respective start sites (18) . (
)Thus for the wild type interpromoter distance of 82 base
pairs a stretch of 10 base pairs or more is expected to be covered by
RNA polymerases bound at P
as well as P
,
creating the possibility of unfavorable contacts between the two
polymerases over this region.
Figure 7:
Model for interference of an RNA
polymerase at P with open complex formation at P
and for relief of the effect by deletion of 10 base pairs. In
this schematic the orientation of the P
and P
promoters is the same as in Fig. 1. Because open complex
formation at P
is very fast, almost every RNA polymerase
forming a complex at P
does so in the context of another
polymerase at P
. The effects of P
occupancy
would be manifest only after RNA polymerase had formed an intermediate
(closed) complex at P
and undergone a conformational
change (represented by the straight arrows). The interference
could be steric or due to occlusion by the RNA polymerase at P
of DNA sites with which the polymerase at P
could
favorably interact. In the former case a 10-base pair reduction in the
distance between the start sites would allow the avoidance of the
unfavorable contacts between the two RNA polymerases if the surface of
RNA polymerase were irregularly shaped. In the latter case the RNA
polymerase at P
would obstruct access to a DNA element
important for P
promoter function (e.g. the
interaction of the C-terminal domain of the
subunit with the UP
element(20) ) on the Wt but not the D10 template (see
text).
For the D5 construct little if any
joint occupancy of the P and P
promoters is
observed. Apparently when the two promoters are rotated by half a turn
with respect to each other by deletion of 5 base pairs between their
-35 regions, increased physical interactions between the two
polymerases prevent them from concurrently being accommodated on the
same DNA fragment. For I5, the two RNA polymerases are oriented as they
would be for D5, but the extra 10-base pair separation between them is
evidently sufficient to allow for their concurrent binding; the I5
construct behaves very similarly to the I10 variant (see Table 1). On I10 the separation of P
and P
is still within the range for which the RNA polymerases bound at
the two promoters might be expected to cover overlapping stretches of
DNA. Steric effects could then play a role here as well, leading to the
observed favorable effect of P
inactivation on
P
function for I10, just as with the wild type template.
The D10 spacing might be unique, resulting in a relative position of
the two polymerases where their surfaces fit to each other so that open
complex formation at the two promoters occurs without interference (as
shown in Fig. 7). An even shorter distance between the two
promoters, as in the lambdoid phage 434, with a 65-base pair distance
between the promoters P
and P
, does not permit
concurrent RNA polymerase binding to the two promoters(10) .
For both I10 and the wild type spacer, the RNA polymerase at P slows down but does not completely inhibit open complex formation
at P
. Such an effect could also be due to the polymerase
at P
interfering with open complex formation at P
by depriving the polymerase at P
from favorable
contacts with certain regions of DNA. In this second model, the
protrusions on the P
-bound RNA polymerase molecules shown
in Fig. 7would be able to contact upstream DNA regions on the
D10 but not the wild type template. Recent results (19, 20) have indicated that the C-terminal domain
(CTD) of the
subunit of RNA polymerase is flexible with respect
to the rest of the enzyme, allowing it to contact AT-rich regions at
various distances upstream of the -35 region. Inspection of the
sequences in Fig. 1shows the presence of a candidate AT-rich
region for the P
promoter, just upstream (with respect to
the P
start site) of the -35 region of
P
. Possibly on the D10 template but not any of the others
investigated, this region is accessible to the
CTD (protrusion in Fig. 7) of the polymerase at P
, despite the
presence of a polymerase at P
. In the case of the wild type
82-base pair spacing between P
and P
(and also
for the 92-base pair spacing of I10), the polymerase at P
would make it difficult for the
CTD of the polymerase at
P
to ``reach'' in far enough to contact the
upstream sequence. However, with the shorter inter promoter distance of
D10, the DNA-binding region of the CTD would be able to overcome steric
barriers imposed by the polymerase at P
. Experiments with
polymerases deleted in the
CTD as well as with templates harboring
sequence alterations in the AT region downstream of the -35
region of P
indicate that such upstream interactions may be
quite important for formation of an open complex at the P
promoter. (
)
The data presented in Table 1hint at a
third way in which on the D10 template an RNA polymerase at P could facilitate open complex formation at P
. With
each of the three techniques a slightly greater rate of open complex
formation at P
was observed for fragments with the D10
spacing in the context of a wild type P
promoter as
compared with fragments with an inactivated P
. Thus for a
72-base pair distance between the start sites, the presence of an RNA
polymerase at P
may actually create a favorable environment
for open complex formation at P
. Such an effect could be
due to favorable interactions between the RNA polymerases at the two
promoters or be transmitted through the DNA itself. In the latter case
one might expect RNA polymerase-induced DNA distortions in the stretch
of DNA between the -35 regions of P
and
P
. Using copper 5-phenyl-1,10-phenanthroline (21) we have searched for but failed to detect such distortions
for RNA polymerase complexes on the templates investigated (Wt, D10,
and I10).