(Received for publication, October 9, 1996, and in revised form, November 29, 1996)
From the Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, Texas 77030
Promoter recognition by RNA polymerase depends
upon its ability to bind to specific DNA sequences. The sigma ()
subunit provides selectivity for transcription initiation by
interacting with the
10 and
35 elements of promoter DNA. Suppressor
mutations in
factor that compensate for specific "down"
substitutions in the promoter have demonstrated that
factor
recognizes certain base pairs of the promoter. Since these suppressors
were only identified for changes at the
12 and
11 positions of the
10 element (
TAAT), the role of the other base pairs of
this region in specifying recognition by
factor remained unclear.
Using a partial polypeptide of
70 carrying regions 2-4,
this report shows that the first three positions of the
10 element
(
12,
11,
10) are important for
factor alone to recognize and
bind to duplex DNA. The
polypeptide also binds to an "extended
10" promoter, even without a
35 element. A mismatch bubble from
10 to +3 is bound regardless of the sequence within the bubble, or
the presence or absence of a
35 element. Unexpectedly, binding to a
mismatch bubble that lacks a
35 hexamer is sensitive to the identity
of the
11 position, but not the
12 position.
Initiation of transcription at bacterial promoters requires the
sigma () subunit of RNA polymerase (1, 2).
factor directs
holoenzyme (
2
) to the recognition sequences
upstream of the transcription start site, and it is required for strand opening on circular DNA (3). A variety of different
subunits is
available for positive regulation of gene expression in response to
environmental stimuli (4). Each
subunit recognizes a distinct DNA
sequence and thereby confers selectivity for initiation upon RNA
polymerase.
The subunits are evolutionarily and functionally related. The
largest family comprises those most similar to the primary
factor
from E. coli,
70 (5). Members of this family
utilize promoters that comprise two hexameric DNA sequences at
approximately
35 and
10 relative to the start point of
transcription (6, 7). For
70, the consensus promoter
consists of TTGACA centered at
35 separated by 17 base pairs from
TATAAT centered at
10. Deviations from consensus usually result in
reduced promoter activity (8, 9). One exception is the "extended
10" promoter, where an additional TGn is found immediately
preceding the
10 hexamer, and a good match to the
35 consensus is
not required for activity (10-15).
During initiation, base pairing of the promoter in the region from
approximately 10 to +2 is disrupted and a transcriptionally competent
open complex is formed (16-19). The precise role of
factor in this
process is not clear, but
factor cross-links between
10 and +1 on
the non-template strand (20, 21). Recently,
70
holoenzyme has been shown to recognize the non-template strand of the
10 sequence in a melted transcription bubble (22). Others have
proposed that
factor stabilizes the single-stranded DNA in the open
complex by interacting with chemical groups in the non-template strand
(23).
Amino acid sequence analysis has revealed four highly conserved regions
within the 70 family (Fig. 1; Refs. 4, 5,
and 24-26). Region 1.1 inhibits DNA binding by the C-terminal DNA
binding domains of
70 in the absence of the core
subunits (
2
), and may physically interact with
amino acids in or near the region 4 helix-turn helix domain at the C
terminus (27, 28). We have recently shown that holoenzyme containing a
70 derivative, which lacks Region 1.1, is significantly
slower in progressing from the initial closed complex to a
strand-separated initially transcribing complex. Additionally, if both
Regions 1.1 and 1.2 are missing from
70, holoenzyme is
arrested in the first closed complex.1
Region 2.4 recognizes the
10 hexamer (29-33), and recent studies suggest that region 2.3 may participate in the DNA melting stage of
initiation (34, 35). Region 4.2 is involved in recognition of the
35
hexamer (27, 29, 36), while region 4.1 has been proposed to contact
certain transcriptional activators during initiation (37-39).
The initial assignment of DNA recognition regions to the polypeptide was accomplished using genetic analyses, starting with point mutations in the
10 or
35 hexamers that severely reduced promoter activity. Suppressor mutations in
factor, which improved utilization of promoter "down" mutations in the
10 hexamer,
localized to region 2.4 (29-32). Interestingly, mutations were only
obtained for suppression of the first two positions of the hexamer.
There are several possible explanations for this observation. The
subunit may only interact with the first two positions of the hexamer,
or the search for suppressor mutations was not saturating, or mutations
that affect recognition of the +1 proximal positions are lethal. Thus,
in this report a direct evaluation of how
70 interacts
with the
10 sequences of the promoter was conducted.
Using a partial polypeptide of 70 and derivatives of the
tac promoter, several aspects of promoter recognition by
in the absence of the core subunits of RNA polymerase have been
addressed. First, the importance of each position in the
10 hexamer
was evaluated. Second, the requirements for interaction with an
extended
10 promoter were examined in the presence and absence of a
35 hexamer. Finally, the role of single-stranded DNA was assessed both alone, and as a component of a mismatch bubble.
Restriction endonucleases, T4 DNA polymerase, T4
DNA ligase, and T4 polynucleotide kinase were from New England Biolabs
or Promega. Taq DNA polymerase was from Perkin Elmer or
Fisher Scientific. [-32P]ATP (3000 Ci/mmol) was from
Amersham Corp. Nitrocellulose filter discs (24 mm) were from Schleicher
& Schuell or Millipore. Oligonucleotides were synthesized by either
Genosys or Bioserve Biotechnology.2
Single-stranded
M13mp19ptac (27) was used as the template for site-directed
mutagenesis. Synthetic oligonucleotides (15-10-mers) were used
according to Kunkel et al. (40) to generate the point mutations at 7 to
12 and
32, as well as the extended
10 version of the tac promoter as shown in Table I.
The mutated M13 derivatives were used as templates for polymerase chain
reaction (PCR)3 amplification of DNA to be
used for competition nitrocellulose filter binding. Non-promoter DNA
(
P) consisted of sequences from the M13 polylinker cloning
region.
|
The "bubble" promoters, bub1/2, bub3/4, bub1/2GC, bub3/4GC, bub3/4-11C, and bub3/4-12G were constructed by annealing two oligonucleotides each 100 nucleotides in length. The annealing mixtures contained 50 µg of each oligonucleotide in 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, and 200 mM NaCl. The mixture was heated to 90 °C in a heat block and allowed to cool slowly to room temperature. The efficiency of annealing was assessed on a 3% Metaphor agarose (FMC) gel.
All of the promoter variants with a deletion of the 35 element, as
well as the variants of ptac-ext-10 with point mutations were constructed using PCR for directed mutagenesis (41) with the
following modifications. Primer III was complementary to primer IV. In
step 1, Primers I plus IV and Primers II plus III were used to produce
a segment of the final DNA product using the next previous generation
of promoter variant as the template. The products of the step 1 PCR
(protocol as described in the following section) were purified using
the Qiaquick PCR purification kit (QIAGEN). A mixture of 5 µl of each
of these products was used in a subsequent PCR step (step 2) with no
additional template for two cycles. Primers I and II were then added
and PCR continued for 35 more cycles. The products were purified either
with the Qiaquick PCR purification kit or, in some cases, if desired
band was agarose gel-isolated, purified with the Qiaquick gel
extraction kit.
Oligonucleotides for
PCR amplification of the ptac derivatives were complementary
to sequences upstream and downstream of ptac such that the
promoter was approximately centered within a 100-base pair length of
DNA. One of the oligonucleotides was 5-end-labeled with
32P as already described and used to generate radioactive
specific DNA (27). Labeled ptac and unlabeled competitor DNA
were synthesized by PCR using 10-15 ng of template (replicative form
of M13mp19ptac derivatives), 50 pmol of each primer, 10 × Taq buffer A from Fisher Scientific, and 2.5 units of either
Ampli-Taq (Perkin Elmer) or Taq (Fisher) DNA
polymerase in a 100-µl reaction. A Perkin Elmer Thermocycler was
programmed for 35 cycles employing 94 °C for denaturation, 55 °C
for annealing, and 72 °C for extension with a time of 1 min for each
segment. The product was purified using the Qiaquick PCR DNA
purification kit (QIAGEN), and visualized on a 3% Metaphor agarose
(FMC) gel.
A fusion protein
between regions 2-4 of 70 and GST, GST
(360), was
used to conduct all filter binding experiments. The construction and
purification of this protein has been described in detail elsewhere
(27). Equilibrium competition binding assays were performed to measure
the reduction in filter retention of
[32P]ptac/GST
(360) complexes as a function
of increasing unlabeled competitor DNA (mutated versions of
ptac). Binding mixtures contained 100 pM
[32P]ptac and various amounts of unlabeled
competitor DNA in binding buffer (10 mM Tris-HCl, pH 7.5, 14 mM potassium acetate, 0.1 mM EDTA, 1 mM dithiothreitol, 0.03% Triton X-100, and 100 µg/ml
bovine serum albumin). GST
(360) was added to 1 nM,
followed by incubation at 37 °C for 20 min. The mixture was filtered
through 24-mm nitrocellulose filters using a Hoefer model FH224V filter
manifold, followed by one wash with wash buffer (10 mM
Tris-HCl, 0.1 mM EDTA, and 1 mM
dithiothreitol). The filters were dried and subjected to liquid
scintillation counting using Ultima gold (Packard). Background DNA
retention was typically 5-10% of the total and was subtracted from
the values obtained for retention of complexes. All data represent the
average of 2-10 duplicate experiments. On any particular day, and for
any set of competitors, ptac and
P were always included as control competitors. Additionally, the same experiments were repeated on different days with different preparations of both DNA and
protein, for every competitor. Thus, the average values presented
account for the variability inherent in the filter binding measurements. As a result, the ptac and
P curves are the
same in each figure. A more detailed discussion of this assay has been published previously (27).
Genetic analysis of function as part of
the holoenzyme complex demonstrated that the first two positions of the
10 hexamer (TATAAT) were important specifically for
promoter recognition by the
subunit of RNA polymerase (29-33). The
remaining four positions are clearly important for holoenzyme function,
since mutations at any of these sites can severely affect promoter
activity (8, 9). These positions may be important for aspects of initiation beyond promoter recognition. For example, cross-linking studies have indicated that the
and
subunits are associated with DNA in the transcription complex (42-44). In this study, the interactions between
factor and each position of the
10 hexamer were examined in the absence of the core subunits of polymerase.
We previously used equilibrium competition binding assays to show that
partial polypeptides of 70, lacking the N-terminal
inhibitory domain, interact preferentially with promoter DNA (27, 45).
In those experiments a 100-base pair DNA fragment carrying the
tac promoter was mixed with increased amounts of unlabeled
competitor DNA, and a fixed amount of
polypeptide. Retention of
·DNA complexes on nitrocellulose filters served as a measure of
DNA binding. A reduction in binding to the labeled DNA by one-half
required addition of either an equivalent amount of unlabeled promoter
DNA or a 5-8-fold excess of unlabeled non-promoter DNA. Since the
actual size of the binding site is unknown, an accurate determination
of selectivity on a per-site basis cannot be ascertained. Thus,
selectivity is at least 5-fold and could be as high as several
hundredfold (27).
Mutations were generated at each of the six positions from 12 to
7
(TATAAT) within the
10 hexamer of the tac promoter (46) to
investigate their relative importance (Table I).
Equivalent substitutions have been shown previously to result in severe
promoter down phenotypes in vivo (8, 9). The ability of the
10 mutant promoters to compete with the unaltered wild type
tac promoter for binding to a partial
70
polypeptide carrying both DNA binding domains fused to glutathione S-transferase, GST
(360), was tested in vitro.
Fig. 2 shows that mutations at
7,
8, or
9 had no
effect on the ability of the unlabeled competitor DNA to compete with
[32P]ptac for binding to GST
(360). These
mutated promoters competed as well as unlabeled ptac (a
specific competitor). Conversely, mutations at positions
10,
11,
and
12 compromised the ability of competitor DNA with these changes
to compete with ptac. Competition by the
10G,
11C, and
12C or
12G DNAs more closely resembled the non-promoter competitor
(
P). These results imply that only the upstream half of the
10
element is required for recognition and binding by the DNA binding
domains of
factor alone.
In previous analyses of competition binding, conditions were
established such that at a 1:1 molar ratio of competitor DNA to
ptac, filter binding was reduced by half (27). The partial factor polypeptide utilized here, GST
(360), has a slightly different composition than those used for competition binding in the
past. It contains all of region 2, in addition to regions 3 and 4, rather than only part of region 2 (GST
(420)). This construction permits an evaluation of binding with the entire conserved region 2 present, which more closely resembles the actual DNA binding components
present in wild type
70. For GST
(360), conditions
that resulted in a 50% reduction in binding at a 1:1 ratio were not
found. The basis for this observation is not clear; however,
GST
(360) does exhibit the expected selectivity for promoter over
non-promoter DNA. The relevant comparison here is not the absolute
amount of DNA needed to compete, but the relative ability of each
mutant promoter DNA to compete compared to promoter (ptac)
and non-promoter (
P) DNA.
Certain prokaryotic promoters can function in the absence
of a 35 element as long as they have the sequence TGnTATAAT, where the addition of TG creates an extended
10 promoter (10-15). Kumar et al. (47) demonstrated that removal of region 4.2 from
70 eliminates the ability of holoenzyme to transcribe
from a promoter with typical
35 and
10 elements, but does not
destroy ability to transcribe from an extended
10 promoter. These
results suggested that perhaps the extended
10 sequence can
compensate for the lack of a
35 element with regard to
factor
binding as well.
Several variants of ptac containing an extended 10
sequence were created. ptac-ext-10 carries a substitution of
G for C at
14 and a substitution of C for G at
13 to generate a
"perfect" extended
10 sequence (see Table I). This promoter
already contained a T at
15. A point mutation from G to A at
32 was
added to the extended
10 to make ptac-ext-10-32A. This
construction combines a promoter down change in the
35 hexamer, which
is known to negatively affect
binding (27), with the extended
10
promoter. Deletion of the
35 hexamer from ptac-ext-10 was
used to generate ptac-ext-10
35. These constructions allow
an assessment of the contribution of the
35 element to GST
(360)
binding to an extended
10 promoter. Equilibrium competition binding
experiments showed that, in general, as long as the promoter contained
the extended
10 sequence it competed as well as ptac for
binding to GST
(360) (Fig. 3A). The presence of a down substitution in the
35 hexamer
(ptac-ext-10-32A) did not affect binding to the extended
10 promoter but did affect binding to ptac. Thus,
factor can recognize and bind to an extended
10 sequence in the
absence of a
35 element, and a point mutation at
32 that normally
disrupts binding by
factor is ineffective in the context of an
extended
10 sequence. This suggests that
makes additional
contacts in or near the
10 region, presumably the TG of the extended
10, that can substitute for loss of the contacts at
35.
Finally, the question of whether point mutations in the 10 hexamer
can be tolerated in the context of an extended
10 sequence and a
deletion of the
35 hexamer was examined. Point mutations were
generated in the ext-10
35 promoter to change each
position of the
10 hexamer to a G:C base pair (see Table I). Fig.
3B shows that changes at positions
8,
9,
11, and
12
do not affect binding to an extended
10 promoter, even in the absence
of a
35 element. Point mutations at
10 and
7 perhaps show a
slightly deleterious effect on binding in this context, but whether
this effect is significant is unclear. An evaluation of transcription initiation by holoenzyme at these promoters may be required to resolve
this point.
In summary, an extended 10 promoter (TGnTATAAT) provides a very
strong recognition signal for the
subunit that can overcome both
the lack of a
35 element as well as point mutations in the
10
element, including mutations at
11 and
12 that seriously affect
binding to ptac without an extended
10 motif. The G at
14 in conjunction with the T at
15 appears to provide a highly effective
factor binding determinant that allows bypass of the typical
35 and
10 determinants. Since ptac normally has
a T at
15, the G at
14 in the extended
10 version may be the key additional contact point for
factor. A double mutation of the G at
14 with another promoter down change may be required to eliminate
binding to the extended
10 sequence.
Open complexes between RNA polymerase
and DNA are characterized by an area of strand separation between
approximately 10 and +2. Cross-linking studies have suggested that
the
subunit is in close proximity to one of the unpaired strands
during initiation (20, 21). One possible explanation for the ability of
factor to bind to DNA with mutations at
7,
8, and
9 is that
these positions are only important once the strands have melted to form an open complex. To test this idea, two bubble promoter constructions were generated for use as competitor DNA. bub1/2 is
identical to ptac from the upstream 5
end to position
11,
and is completely base-paired along this segment. From
10 to +2,
however, the two strands are non-complementary, with both corresponding
to the non-template strand (see Table I). At position +3, base pairing is resumed toward the downstream end of the fragment. This creates a
bubble in the promoter that mimics the transcription bubble formed by
RNA polymerase holoenzyme during transcription initiation. bub3/4 is similar to bub1/2, except the unpaired
segment contains the sequence corresponding to the template strand of
ptac. Similar bubbles have been used by others for
transcriptional analysis of RNA polymerase (48, 49).
Equilibrium competition experiments with GST(360) using mismatch
bubble DNAs as competitors demonstrated that
factor binds as well
to bubble DNA as to duplex ptac DNA (Fig.
4A). DNA with the template
(bub3/4) or non-template (bub1/2) strand in the
bubble was bound similarly. These results agree with the idea that
positions
11 and
12 may be the most critical sites for
factor
recognition of base-paired promoter DNA, as predicted by earlier
genetic suppression studies. To determine if GST
(360) might be
binding to the bubble constructs merely because the unpaired sequence
in both bub1/2 and bub3/4 is AT-rich, three
changes were made at
10,
9, and
7 of both competitors to generate
bubbles with higher GC content (see Table I). These DNAs,
bub1/2GC and bub3/4GC, also competed well with
ptac for binding (Fig. 4A). Thus it appears that
once the DNA has been strand-separated to form a bubble, the sequence within the bubble is not a parameter in determining the ability of
factor to bind, and unlike duplex DNA, substitution at the
10
position is tolerated.
To further examine the question of whether the base-paired 11 and
12 positions in the
10 hexamer are important in the context of a
bubble, point mutations at
11 from A to C and at
12 from T to G
were combined with bub3/4. These changes are known to weaken promoter strength and disrupt binding by regions 2-4 to duplex promoter DNA. Surprisingly, both bub3/4-11C and
bub3/4-12G competed as well as ptac for binding
(Fig. 4B), indicating that in the context of a bubble, the
11 and
12 positions are not crucial for binding by
factor, even
though these mutations have been shown to disrupt binding to fully
double-stranded DNA (27).
If none of the 10 hexamer positions are important for GST
(360) to
bind to a bubble promoter, then one explanation is that the
35
element provides an "anchoring" point that allows
factor to
bind selectively. The role of the
35 element, in the context of a
bubble was addressed by constructing several additional promoter variants. The
35 hexamers of bub1/2, bub3/4,
bub3/4-11C, and bub3/4-12G were deleted to
generate bub1/2
35, bub3/4
35,
bub3/4-11C
35, and bub3/4-12G
35. Deletion
of the
35 element in the context of either bubble
(bub1/2
35 or bub3/4
35) did not affect
binding (Fig. 4B). Thus, the presence of the bubble can
compensate for a lack of a
35 sequence. Interestingly,
bub3/4-12G
35 still competed for binding to GST
(360),
while bub3/4-11C
35 did not. Thus, the
11 position
cannot tolerate substitution in combination with a bubble and the
removal of the
35 element. On the other hand, the identity of the
12 base pair in this context is flexible. In summary, GST
(360)
binds well to any of the bubble constructs with the only requirement
being a favorable
11 base pair, providing the
35 element is
absent.
Potential interactions between factor and single-stranded
DNA were probed using the bub1, bub2,
bub3, and bub4 single-stranded oligonucleotides
as competitors for GST
(360) binding to double-stranded ptac. As shown in Fig. 5, none of the
single-stranded DNAs was able to compete for binding with
ptac. Irrespective of the sequence in the
10 region, each
of these DNAs behaved like the non-promoter competitor,
P. Thus, the
subunit prefers to interact with DNA that is either completely
base-paired or partially unpaired to resemble a transcription
bubble.
The role of the subunit of RNA polymerase in directing
promoter recognition is well established. Mutations at any one of the
six positions of either the
10 or the
35 promoter element can
dramatically affect promoter utilization by holoenzyme (8, 9). The
subunit is largely responsible for direct interactions at both the
10
and
35 sequences. Like holoenzyme, a partial
polypeptide carrying
both DNA binding domains is sensitive to point mutations in either the
35 or
10 hexamer, despite the fact that each DNA binding domain
functions independently when present on separate polypeptides (27). It
has therefore been proposed that each DNA binding domain must contact
the appropriate DNA site concomitantly to form a stable protein-DNA
complex (45).
Genetic analysis of function previously resulted in the
identification of several mutations in region 2.4 that suppressed promoter down changes at the first two positions of the
10 hexamer (29-33). No mutations in
were isolated that suppressed promoter down changes in the last four positions (
10 to
7 for
ptac). Since partial polypeptides of
70, in
the absence of the core subunits, can be used to evaluate binding
specificity (27), it became possible to directly assess promoter
recognition by
at the
10 element. The results presented here
address the sequence requirements in the
10 region for
binding,
the contribution of extended
10 sequences to
binding, and the
potential interactions between
and unpaired bases in the
10
hexamer.
Base pair changes away from consensus at positions 10,
11, and
12
of ptac (TATAAT) reduce the ability of a
polypeptide carrying regions 2-4 of
70, GST
(360), to
bind to these promoters in vitro. This is consistent with
the reduced transcriptional activity of holoenzyme at similarly mutated
promoters in vivo. In contrast, mutations at positions
9,
8, and
7, which also compromise holoenzyme activity in
vivo (9), do not affect the ability of GST
(360) to bind
in vitro. Similar binding selectivity was observed for a
polypeptide containing region 2 only of
70 (data not
shown), and thus it is unlikely that the GST moiety is affecting the
behavior of the
portion of the fusion polypeptide.
A variant of the 10 consensus has also been characterized for
GST
(360) binding in which TGn appears immediately upstream of the
first position of the hexamer. This extended
10 sequence can
alleviate the need for a
35 consensus (13, 15, 47) or can strengthen
promoters that already contain a
35 element (14, 50, 51). One
explanation for the apparently improved interactions between an
extended
10 promoter and RNA polymerase is that perhaps the
subunit makes additional contacts with the DNA. This is supported by
the observation that a C-terminal truncation of
70 that
removes region 4.2, the
35 recognition domain, maintains its ability
to facilitate transcription initiation at an extended
10 promoter but
not a standard promoter (47).
In the experiments presented here, GST(360) binds equally well to a
normal ptac promoter, ptac lacking a
35 element
but containing an extended
10 sequence (ptac-ext-10
35),
or ptac with a down mutation in the
35 element combined
with an extended
10 sequence (ptac-ext-10-32A). Although
these observations are consistent with the behavior of holoenzyme at
similar promoters, this is the first demonstration that the
subunit
is the primary determinant for this mode of recognition. The binding
behavior of GST
(360) at ptac-ext-10
35 variants with
single substitutions in the
10 hexamer is not as straightforward.
None of the point mutants creates a promoter that GST
(360) fails to
recognize to some extent, since binding is better in all cases than to
non-promoter DNA (
P). Overall,
factor may be involved in
particularly strong interactions at the TG upstream of the
10 in the
extended
10 promoter, and needs neither a
35 nor a perfect
10
hexamer to bind. Since the original ptac promoter has a T at
15, the G added at
14, to make the extended
10, appears to be the
critical site for additional contacts between
and the extended
10
sequence. These additional contacts alleviate the requirement for
contacts at the
35 element, which are usually required for binding to ptac. These results imply that the extended
10 promoter is
a more powerful element in directing transcription than has previously been realized.
All of the positions of the 10 hexamer are important for optimal
promoter usage by holoenzyme (9, 52). Combined with the fact that
positions
9 to
7 appear to be unimportant for GST
(360) binding
on double-stranded DNA, one possibility is that
actually interacts
with single-stranded DNA within the transcription bubble. In the
context of holoenzyme, these positions may not be important for initial
binding of polymerase to the promoter, but instead may be required for
isomerization or strand separation to form a stable open complex during
transcription initiation. Several studies have implicated region 2.3 of
in the process of strand melting, but the precise mechanism of
involvement remains obscure (34, 35, 53).
Using DNA with a mismatch bubble extending from 10 to +3, the results
presented here show that
can bind to these promoters when the
template strand, the non-template strand, or a GC-rich version of
either the template or non-template strand constitutes the sequence in
the bubble. Thus, GST
(360) does not seem to discriminate between
base pairs in the strand-separated promoter region, including the
10
position, which is important for binding to duplex DNA. This is in
contrast to recent studies indicating that holoenzyme recognizes
unpaired bases in a
PR
heteroduplex promoter at
10 of
the non-template strand (22). Additionally, a proteolytic fragment of
70 containing amino acids 114-448 (C-terminal half of
region 1.2 through most of region 2) forms a complex with the core
subunits of polymerase that then binds to single-stranded DNA, which is comprised of the
10 sequence of the non-template
strand.4 However, this
fragment does
not bind to DNA in the absence of core, leading to a proposal that
conformational changes in the fragment upon core binding allow regions
2.3 and 2.4 to interact with the non-template single-stranded DNA in
the open complex. Thus for GST
(360), it is also very likely that in
the absence of core, the conformation of the polypeptide does not favor
specific interactions with single-stranded DNA in the
10 hexamer. The
subunit appears to need to be part of a complete initiation complex
in order to recognize the
10 base pairs on the non-template strand in
the natural transcription bubble.
Unexpectedly, GST(360) still binds to a mismatch bubble with the
11C or
12G mutation, whereas the same change alleviates binding of
double-stranded DNA. This predicts that
may have a high nonspecific
affinity for any single-stranded DNA. However, GST
(360) does not
bind to completely single-stranded DNA, regardless of the identity of
the strand or the sequence in the
10 region. Finally, the importance
of the
35 hexamer in the context of a mismatch bubble was evaluated.
Surprisingly, deletion of the
35 elements of either bub1/2
or bub3/4 has no effect on binding by GST
(360). The
bub3/4
35 promoter can even tolerate a promoter down point
mutation at
12 and still compete for binding. However, a
11
promoter down change causes the bub3/4
35 DNA to become a
poor substrate for binding. Since there was no evidence for the
sequence within the bubble affecting binding, bub1/2
35
variants with
11 and
12 substitutions were not tested.
The subunit appears to recognize the
10 hexamer in two different
contexts, in the presence of the core subunits as holoenzyme, and in
the absence of core. The results presented here are consistent with the
following scenario for recognition of promoter DNA by
factor.
First, holoenzyme is sensitive to changes at all six positions of the
10 and also requires a
35 element to firmly "anchor" the
protein to the promoter. At an extended
10 promoter, holoenzyme makes
extra contacts with the
10 region of the DNA, eliminating the need
for a
35 anchor.
factor, in the absence of core, appears to be
involved in interactions with the first three positions of the
10
element (TATAAT) in binding of double-stranded DNA. The
identity and single or double-stranded character of the remaining three
positions (
7 to
9) is not a factor in recognition by
as long as
the
35 element is present. Second, the presence of an extended
10
very effectively replaces the requirement for a
35 element presumably
by allowing
to make additional promoter contacts that stabilize the
complex. While the substitution of an extended
10 for a poor or
absent
35 has been invoked in the past to explain promoter activity, this is the first demonstration that the
subunit is primarily responsible for utilization of the extended
10 motif.
Finally, while does not bind to single-stranded DNA, it has an
expectedly high affinity for DNA that is primarily double-stranded but
that contains a mismatch bubble in the
10 sequence. The specificity of
for specific bases in the single-stranded transcription bubble that was demonstrated by Roberts and Roberts (22) was not observed using a partial polypeptide of
alone, in agreement with the behavior of a proteolytic fragment of
.4 It appears
likely that interactions between
and core dictate this mode of
recognition. During transcription initiation,
participates in steps
beyond initial promoter binding. It has recently become clear the
Regions 1.1 and 1.2 are involved in transitions following promoter
binding.1 Thus, the conformation and positioning of
on
the DNA may change at each step of initiation. Since the behavior of
holoenzyme at the various promoters utilized here, for either DNA
binding or transcription initiation, has not been analyzed, the
contribution of the core subunits to utilization of these promoters
remains to be determined.
The recognition of promoters by RNA polymerase is a complex subject.
There are, of course, promoters that contain a weak match to the 10
consensus yet function in transcription. However, many parameters other
than the sequence of the hexamers come into play in determining the
overall efficiency of a particular promoter (54). These include the DNA
topology in the region, participation of activators or other
DNA-binding proteins, and interaction of the
subunit of polymerase
with DNA upstream of the promoter. Interestingly, recent studies of the
promoter recognition properties of the stationary phase
,
38 or
S, indicate that the
10 hexamer
but not the
35 is essential for transcription initiation from
S-dependent promoters (55-57). It is clear
that the
10 hexamer plays an important role in specifying
transcriptional start sites and that the
subunit of RNA polymerase
has a primary role in utilization of specific sequences at that region
of the promoter.
I thank Dr. C. Gross, in whose laboratory this work began, for advice, encouragement, and critical reading of the manuscript; Dr. P. deHaseth for suggestions for making mismatch bubble DNA; and the members of my laboratory for helpful discussions.