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INTRODUCTION |
Promoter sequences involved in
recognition by Escherichia coli RNA polymerase (RNAP) were
identified from comparisons of a large number of known promoters and
from mutational analyses (28, 29, 38, 62). These sequences,
the
10 and
35 hexamers (5' TATAAT 3' and 5'TTGACA
3', respectively), are recognized by the
70
subunit of RNAP (11). The strength of a promoter correlates generally with its degree of identity to these sequences and with the
length of the spacer between them (the homology score
[42]), although exceptions to this rule have been
described (7, 26).
It was proposed more than 10 years ago that optimal transcription
activity could be achieved by different combinations of promoter
elements, including not only the
10 and
35 hexamers, but also
upstream and downstream regions (7). In accord with this
suggestion, RNAP protects regions both upstream and downstream of the
10 and
35 hexamers in footprints (8, 45, 47, 56), and A+T-rich sequences upstream of the
35 hexamer in several E. coli or Bacillus subtilis promoters were found to
increase transcription in vitro in the absence of accessory proteins
(3, 19, 31, 37, 39, 50, 54). Phased A-tracts inserted upstream of the
35 region in various promoter constructs were also
shown to increase transcription (6, 12, 24).
The A+T-rich region upstream of
40 in the rRNA promoter
rrnB P1, the UP element, increases transcription 30- to
70-fold by binding the RNAP
subunit (13, 50, 53). A
consensus UP element sequence was determined by using in vitro
selection for upstream sequences that promote rapid RNAP binding to the
rrnB P1 promoter, followed by in vivo screening for high
promoter activity. The consensus UP element consists of alternating A-
and T-tracts (13). UP elements matching the consensus
increased promoter activity as much as 326-fold, about 5-fold more than
the wild-type rrnB P1 UP element. UP elements were also
identified in other promoters, for example, the flagellin
(hag) promoter of B. subtilis (18),
the PL2 promoter of phage lambda (25), and the
Pe promoter of phage Mu (61), although the
effects of these elements were not as large as that of rrnB
P1. UP elements also function in promoters recognized by RNAP
holoenzymes with alternate
factors (18).
UP elements are not as highly conserved as the
10 and
35 elements
and were not described in studies comparing the large sets of E. coli promoters used to define the consensus hexamers (28, 29,
38). However, A+T-rich sequences were identified as a prominent
feature of a subset of E. coli promoters (the
44 motif
[23]), and a recent E. coli promoter
analysis (48) identified two A+T-rich regions at upstream
positions corresponding to those crucial for UP element function
(14). A+T-rich upstream sequences were also identified in
compilations of B. subtilis and Clostridium promoters (27, 30).
We have proposed that UP elements may be a recognition feature in many
bacterial promoters (13, 53), but in most promoters, the
role of upstream sequences has not been evaluated experimentally. Therefore, in this paper, we have examined the role of upstream sequences from six promoters (rrnB P2, rrnD P1,
RNA II, merT, lac, and
pR). We find that several of the sequences
function as UP elements and that their effects on promoter activity
differ, correlating generally with the degree of similarity to the UP element consensus sequence. These results support the model that bacterial promoters consist of at least three modules, not just
10
and
35 elements. We also show that upstream protection in footprints is not a reliable indicator of UP element function.
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MATERIALS AND METHODS |
Strains and plasmids.
The strains and plasmids used in this
study are listed in Table 1. Single-copy
promoter-lacZ fusions were constructed by inserting
promoters as EcoRI-HindIII fragments into
either of two phage lambda lacZ fusion vectors (system I for
promoters with higher activities or system II for promoters with lower
activities [50]). Monolysogenic strains were
identified by comparison of the
-galactosidase activities of several
independent candidates and by a PCR test (13). System I
phages carrying
pR promoter-lacZ fusions contain the immunity region of phage 21, introduced from
i21 phages in vegetative crosses. System II
phages all contain the immunity 21 region. Plasmids used for in vitro
transcription were derivatives of pRLG770 (52) and contained
EcoRI-HindIII promoter fragments inserted
~170 bp upstream of an rrnB T1 terminator.
EcoRI-HindIII promoter-containing fragments
were obtained by PCR from plasmid templates containing other
derivatives of the same promoter, except as noted. Upstream primers for
PCR contained an EcoRI site and the upstream 25 to 30 nucleotides of the promoter. The downstream primer (RLG1620) contained
vector pRLG770 sequence (5'-GCGCTACGGCGTTTCACTTC-3') about
40 bp downstream of the HindIII site for insertion of
the promoter fragment. rrnD P1 promoter fragments were
obtained by PCR from plasmid pRLG3246 [rrnD P1 (
61 to
+10)] (22). The
pR (
60 to +20)
promoter fragment was obtained from EcoRI and
HindIII digestion of pBR80 (58) and contained
about 120 bp of pBluescript vector sequence both upstream and
downstream of the promoter.
Hybrid promoters containing upstream elements from different sources
fused to either the lac or the lacUV5 core
promoter at position
37 were constructed by PCR with plasmids
pRLG1821 (50) and pRLG593 (53) as templates.
Upstream primers contained an EcoRI site, the desired UP
element sequence, and lac core promoter sequence from
37
to
17. The downstream primer was RLG1620 (see above). The upstream
sequences of the hybrid-lac promoters are shown in Fig. 5.
Promoter activity determinations.
Promoter activities were
determined in vivo by measurement of
-galactosidase activities in
strains lysogenic for
carrying the promoter-lacZ
fusions. Cultures were grown for 4 or more generations in Luria-Bertani
medium at 30°C (for system I lysogens) or at 37°C (for system II
lysogens), and mid-logarithmic-phase cells were used to measure
activities as described previously (41).
In vitro transcription was carried out essentially as described
previously (52, 53) in reaction mixtures containing 50 ng of
supercoiled plasmid DNA template, 10 mM Tris-Cl (pH 7.9), 10 mM
MgCl2, 1 mM dithiothreitol, and 100 µg of bovine serum
albumin per ml. Reaction mixtures also contained either 30 mM KCl (see Fig. 2B and C and 3) or 50 mM KCl (Fig. 2A) and the following nucleoside triphosphate (NTP) concentrations: Fig. 2A, 500 µM ATP, 50 µM GTP and UTP, 10 µM CTP, and [
-32P]CTP; Fig. 2B,
500 µM ATP, 50 µM UTP, 10 µM GTP and CTP, and [
-32P]GTP; Fig. 2C, 100 µM ATP, CTP, and GTP, 10 µM UTP, and [
-32P]UTP; Fig. 3, 400 µM ATP, 100 µM GTP and UTP, 10 µM CTP, and [
-32P]CTP.
[
-32P]NTPs were from DuPont and were used at about 5 µCi per reaction.
DNase I footprinting.
DNA fragments were prepared by
digestion of plasmid DNAs (pRLG2227, pRLG947, pRLG946, pRLG945,
pRLG943, and pRLG593 [Table 1]) with HindIII (at
promoter position +40), labelling of the top (nontemplate) strand with
Sequenase (Amersham) and [
-32P]dATP (DuPont), and
further digestion with AatII (77 bp upstream of the
EcoRI site at the upstream end of the promoter). Fragments were gel isolated and purified and concentrated with Elutip D columns
(Schleicher & Schuell). RNAP or
subunit complexes with promoter
fragments (0.5 nM) were formed at 26°C in a mixture of 30 mM KCl, 40 mM Tris-acetate (pH 7.9), 10 mM MgCl2, 10% glycerol, and
100 µg of bovine serum albumin per ml and were digested with DNase I
at 2 µg/ml for 30 s. Heparin (10 µg/ml) was added to
RNAP-promoter complexes prior to DNAse I digestion. Processing and
electrophoresis of samples were performed as described previously
(52, 53).
RNAPs and
subunit.
Wild-type,
265A, and 
235
RNAPs were obtained from A. Ishihama (53) or were
reconstituted from purified subunits as described previously
(21).
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RESULTS |
We tested whether a series of promoters contained UP elements by
(i) measuring the effects of their upstream sequences in vivo with
promoter-lacZ fusions, (ii) determining the effects of their
upstream sequences in vitro with wild-type and
-mutant RNAPs, and
(iii) characterizing the interactions between the upstream sequences
and RNAP or purified
subunit in vitro by DNase I footprinting. We were particularly interested in whether the effects of different UP
elements on transcription would correlate with the number of sequence
matches to the recently defined UP element consensus (13)
and whether footprints would be a reliable indicator of the
presence or absence of an UP element.
Effects of upstream sequences on transcription in vivo.
The
effects of upstream sequences on promoter activity in vivo were
determined for four promoters, chosen because previous in vitro data
indicated that their upstream sequences might increase their activities
in vivo. For two of the promoters, upstream sequences were protected by
RNAP in footprints (
pR and
lac [8, 34]), and for the other two
promoters, upstream regions stimulated promoter activity in vitro
(rrnB P2 and rrnD P1 [53, 54]).
Derivatives of each promoter containing either native or substituted
upstream sequences (Fig. 1) were fused to
lacZ, and their activities were determined by measuring
-galactosidase levels in strains containing chromosomal copies of
the fusion constructs (Table 2). For each of the promoters, the derivative with native upstream sequences had
more activity than the derivative with substituted sequences, although
the magnitudes of the effects were very different. The rrnD
P1 upstream sequence increased transcription dramatically (approximately 90-fold), even more than the previously characterized rrnB P1 UP element (Table 2) (13, 50). The
rrnB P2 upstream sequence also increased transcription, but
to a much lesser extent (3.3-fold), while the
pR and lac upstream sequences had
only very small effects (1.7- and 1.5-fold, respectively). The
lac promoter sequences responsible for the 1.5-fold effect
appeared to include the region upstream of
47, since lac
promoters with upstream endpoints of
40 or
47 had slightly lower
activities than the
60 derivative.

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FIG. 1.
Sequences of derivatives of four E. coli
promoters, rrnD P1, rrnB P2, pR, and lac, containing either native
or substituted upstream sequence. Promoters are designated by the
position of the upstream-most native position (e.g., rrnD P1
60 has native sequence to 60). Native sequences are represented by
uppercase letters, and substituted sequences are represented by
lowercase letters. EcoRI sites at the junction of promoter
and vector sequences are italicized. The 10 and 35 hexamers are
shown in boldface. Substituted sequences for rrnD P1 41,
rrnB P2 39, pR 42, and
lac 47 are from the lambda phage vector into which they
were cloned for in vivo activity measurement (Table 1). For the
lac 40 promoter, the substituted sequence was the SUB
sequence (50). The phage vector sequences and the SUB
sequence were previously characterized in the context of the
rrnB P1 promoter and did not affect transcription (50,
53).
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Upstream sequences affect transcription directly.
Upstream
sequence effects on transcription in vivo could result from direct
interactions with RNAP or from effects of transcription factors. To
distinguish between these possibilities, upstream sequence function was
characterized in vitro (Fig. 2).
Transcription of the rrnD P1 promoter containing its native
upstream sequence (to
60) was much stronger than that of the promoter
with native sequence only to
41 (Fig. 2A, lanes 1 to 4), indicating a
direct effect of the upstream region on RNAP. This effect was not seen with a mutant RNAP defective in UP element recognition (
R265A RNAP
[21]) (Fig. 2A, lanes 5 to 8), indicating that the
rrnD P1
41 to
60 sequence functions as an UP element.
This rrnD P1 upstream region corresponds to the position of
the rrnB P1 UP element (50) and partially
overlaps a region in rrnD P1 previously found to stimulate
its function in vitro (
50 to
89 [54]). The
rrnD P1 UP element had a greater effect than the
rrnB P1 UP element in vitro (51), consistent with
their relative effects in vivo (Table 2). Under these in vitro
conditions, the effects were not as large as those observed in vivo
(Table 2 and Fig. 2A) (13, 53). However, for rrnB
P1, larger effects of the UP element were observed at higher-salt and
lower-RNAP concentrations, and kinetic studies have revealed in vitro
effects similar to those observed in vivo (13, 50). We
expect that a similar situation would be true for rrnD P1.

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FIG. 2.
In vitro transcription of promoters containing or
lacking native upstream sequences. (A) rrnD P1 promoters
with upstream endpoints of 60 or 41 (plasmid templates pRLG3266 and
pRLG3267) transcribed with 0.5 nM wild-type (WT) RNAP (lanes 1 to 4) or
0.5 nM R265A mutant RNAP (lanes 5 to 8). (B) pR promoters with upstream endpoints of 61 or
42 (plasmid templates pRLG2229 and pRLG936) transcribed with 1 nM
wild-type RNAP. (C) RNA II promoters with upstream endpoints of 150
or 42 (plasmid templates pRLG934 and pRLG938) transcribed with 2 nM
wild-type RNAP. In each experiment, transcripts were separated on a 5%
acrylamide-7 M urea gel, and the promoter-specific and the
vector-encoded RNA I transcripts are indicated.
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The rrnB P2 upstream sequence examined above (Table 2) was
previously shown to increase transcription in vitro in the absence of
additional factors and to require the
subunit C-terminal domain
(
CTD) for its effect (53). Thus, we conclude that
rrnB P2 has an UP element with a modest degree of function
in vivo.
The
pR and lac upstream sequences
had very little effect on transcription in vivo. Nevertheless, we
examined
pR in vitro by using derivatives
with native (to
61) or substituted (to
42) upstream sequences to
distinguish whether possible inhibitory factors might have obscured
detection of stimulatory effects in vivo (Fig. 2B). No effect of the
native upstream sequence was observed. We did not examine the
lac upstream sequences in vitro, since this promoter's
activity in the absence of the activator protein, CRP, was too low to
be quantified by this assay (see below).
An additional promoter, RNA II, which makes the primer for ColE1
plasmid replication, was also included in our study. This promoter
(with native upstream sequences to
150) was not efficiently transcribed by
-mutant RNAP in previous transcription experiments, suggesting that it might contain an UP element (53). To
further characterize its upstream sequences, we constructed an
additional promoter derivative with sequences extending only to
42
and compared the activities of the two promoters in vitro (Fig. 2C).
The native upstream sequences increased transcription by wild-type RNAP
in the absence of other protein factors, indicating that the RNA II
upstream sequence functions directly. Although the RNA II upstream sequence was not examined in the context of its own promoter in vivo,
it stimulated the activity of the lac core promoter (see below). We also observed that expression of plasmid-encoded
subunits defective in DNA binding and UP element function reduced the
maintenance of ColE1 plasmid derivatives, suggesting that the RNA II UP
element does function to stimulate its promoter in vivo
(21).
Relative strengths of different UP elements in the context of the
same core promoter.
The widely varying effects of different
upstream sequences on transcription (Table 2) could reflect differences
in their interactions with the
subunit and/or differences in the
capacity of the core promoters to respond to an UP element (i.e., core promoter mechanisms could be limited to different extents by a step or
steps affected by UP elements). To compare directly the relative
strengths of several upstream sequences, their effects on the same core
promoter were determined with hybrid promoters. The upstream
sequences from rrnB P2, RNA II,
pR, and merT were fused to the
lac core promoter. The merT sequence was included in the study, since it was protected by RNAP in footprinting
experiments (46). The lac core promoter was used
for the hybrid promoter constructs, since we showed previously that it
responds to the rrnB P1 UP element in an rrnB
P1-lac hybrid (50) (Table
3).
The activities of the hybrid promoters were compared in vivo with that
of the lac promoter without an UP element (
40
lac [Table 3]). The rrnB P1 UP element had the
largest effect, increasing lac transcription ~33-fold,
consistent with previous observations (50). The
rrnB P2, RNA II, and merT sequences increased
transcription 12.8-, 4.9-, and 2.0-fold, respectively, while the
pR upstream region did not increase
lac promoter activity significantly.
The rrnB P1, rrnB P2, RNA II, and merT
upstream sequences affected lac promoter activity directly,
since the hybrid promoters had greater activity in vitro than the
lac promoter without an UP element (Fig.
3A) (50). Transcription from
the lac core promoter (
40 lac) and from
pR-lac was not detectable (Fig. 3A,
lanes 1 and 2) (51). Thus, the activities of the hybrid
promoters in vitro were consistent with their relative activities in
vivo: rrnB P1-lac > rrnB
P2-lac > RNA II-lac > merT-lac >
pR-lac (Table 2 and Fig. 3A).
The stimulation of lac promoter activity by the upstream
sequences in vitro was dependent upon the DNA binding function of the
RNAP
subunit, since no transcription from the hybrid promoters was
observed with RNAP lacking the
CTD (
235 RNAP [Fig. 3B, lanes 1 to 5]), although the mutant enzyme was proficient in transcription of
lacUV5 (lane 6).

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FIG. 3.
In vitro transcription of lac and
hybrid-lac promoters with wild-type RNAP (A) or  235
mutant RNAP (B). The upstream sequences of the hybrid promoters and
their junction with the lac core promoter (at position 37)
are shown in Fig. 5. Transcripts were separated on 5% acrylamide-7 M
urea gels, and hybrid promoter, lacUV5, and vector-encoded
promoter RNA I transcripts are indicated. Duplicate samples are shown
in panel A. The RNAP concentrations were 2 nM (A) and 8 nM (B). Plasmid
templates for transcription were as follows: lac, pRLG1821;
rrnB P1-lac, pRLG1820; RNA II-lac,
pRLG940; merT-lac, pRLG941; rrnB
P2-lac, pRLG942; and lacUV5, pRLG593.
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Interaction of upstream elements with the RNAP
subunit.
The experiments presented above identified a requirement for the DNA
binding function of the
subunit for upstream sequence function,
suggesting that as with the rrnB P1 UP element,
CTD interacts directly with these sequences. We confirmed this conclusion by footprinting with hybrid promoters in which the upstream
sequences were fused to the lacUV5 core promoter (Fig.
4). (lacUV5, a promoter with a
2-bp substitution mutation in lac that creates a consensus
10 hexamer, was used to improve promoter occupancy by RNAP; we assumed that the lacUV5 mutation did not affect the
subunit-UP element interaction directly.)

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FIG. 4.
DNase I footprints of hybrid-lacUV5
promoters containing various different upstream sequences (A to E) and
of lacUV5 with its native upstream sequence (F). In each
case, the top (nontemplate) strand was radiolabelled at position +40.
A+G, sequence markers; 0, no RNAP; WT, wild-type RNAP (40 nM); 235,
 235 mutant RNAP (40 nM). The core promoter-protected regions are
indicated by a thin line, and upstream regions protected by wild-type
RNAP, but not by  235 mutant RNAP, are indicated with a thick
line. Positions in the upstream regions hypersensitive to DNase I are
indicated by asterisks. In panel B, rrnB
P2-lacUV5 was also tested in the presence of purified subunit (2 to 8 µM, lanes 4 to 6). The region protected by the subunit is indicated with a hatched bar.
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Protection of the rrnB P1 UP element when fused to the
lacUV5 promoter (Fig. 4A) was comparable to its protection
in the context of its own core promoter (53). The A+T-rich
UP element was cleaved inefficiently by DNase I, as expected (lane 2)
(53, 57), but several protected positions were detected in
the presence of wild-type RNAP (lane 3). This protection was not
observed with RNAP lacking the
CTD (lane 4).
Protection was also observed upstream of
40 in wild-type RNAP
footprints of each of the other hybrid promoters and of
lacUV5 with its native upstream sequence (Fig. 4B, lane 2, and C to F, lanes 3). In each case, upstream sequence protection
required the
CTD (Fig. 4B, lane 1, and C to F, lanes 4). Protection
in three of the promoters occurred in two short regions
(approximately
41 to
43 and
50 to
53) that correspond to the
proximal and distal positions protected against hydroxyl radical
cleavage in the rrnB P1 UP element (lacUV5, RNA
II-lacUV5, and rrnB P2-lacUV5) (Fig.
4B, C, and F) (13, 45). In the merT and
pR upstream sequences, protection occurred in
the distal region (
51 to
53), but was not as evident in the
proximal region (
41 to
43). An additional partially protected
region (around
60) occurred in some of the footprints (e.g.,
lacUV5 and
pR-lacUV5).
In each promoter, positions around
44 to
48 were accessible and, in some cases, were hypersensitive to DNase I (see Fig.
5 for sequences). The core region of each
hybrid promoter (Fig. 4) was protected by both the wild-type and

235 RNAPs, and as observed previously for lacUV5
(34), contained sites in the spacer region (at
24 and
25) that were hypersensitive to DNase I cleavage.

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FIG. 5.
Sequences of promoter upstream regions compared to the
UP element consensus (from reference 13). Upstream
sequences shown are those in the hybrid-lac promoters
(Tables 1 and 3 [see Materials and Methods]), except for
rrnD P1, which is from the rrnD 60 promoter
(Fig. 1). Matches to the consensus are indicated in uppercase and
boldface type. Vector-derived sequences are underlined, and native
upstream sequences not matching the consensus are in lowercase type.
The 35 hexamer regions are represented by open boxes. Positions
accessible or hypersensitive to DNase I cleavage (Fig. 4) are indicated
by arrows. The relative activities of the upstream sequences represent
their function in the context of the hybrid-lac promoters
(Table 3), except for rrnD P1 (indicated with an asterisk),
which was determined in the context of its own core promoter (Table
2).
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We also tested the binding of purified
subunit to the three UP
elements with moderate effects on transcription, rrnB P2, RNA II, and merT. Specific protection of the rrnB
P2 UP element was observed from ~
36 to
53 (Fig. 4B, lane 6).
Approximately fourfold-higher
subunit concentrations (4 to 8 µM)
were required for protection of the rrnB P2 UP element than
for protection of the rrnB P1 UP element in the same
experiment (51, 53). At higher concentrations of
subunit, the rrnB P2-protected region extended further
upstream, to approximately
62, similar to the boundary observed with
the rrnB P1 UP element (51, 53). Protection of
the merT UP element region was observed at ~8 µM
subunit, while specific protection of the RNA II upstream region was
not observed (51).
 |
DISCUSSION |
UP elements of different strengths.
We identified upstream
sequences in several E. coli promoters that had the
characteristics of UP elements: they increased transcription in vivo as
well as in vitro in the absence of factors besides RNAP, and their
function depended on their interaction with the
subunit of RNAP.
Effects of the different upstream sequences examined here varied
widely, by a factor of almost 100. We arbitrarily define as UP elements
only those sequences that increased transcription twofold or more in
vivo. We do not define the lac and
pR upstream sequences as UP elements, since they affected transcription in vivo only slightly, these effects were defined relative to the function of an arbitrary "neutral"
sequence, and their effects were not observed in vitro. Although
they did not significantly affect promoter function, the lac
and
pR sequences were protected in
footprints with wild-type RNAP, and this protection was
CTD
dependent (Fig. 4). Thus, footprint protection of an upstream
sequence is not sufficient to define a functioning UP element in the
absence of other evidence.
The negligible effect of the lac upstream sequence on
promoter activity is consistent with previous observations on the
effects of promoter substitution mutations in this region
(16) and of
subunit mutations on lacUV5
activity in vitro (53). Although
CTD interactions with
lac upstream DNA are insufficient to increase transcription
in the absence of CRP, they have been observed in footprinting
experiments performed in the presence of RNAP and CRP (34)
and appear to play a role in activator-dependent transcription by
contributing to the overall stability of the activator-RNAP-promoter complex (9, 21, 59).
Similarity to consensus as a predictor of UP element function.
Effects of upstream sequences on transcription correlate generally with
the extent of their similarity to the consensus UP element (Fig. 5).
The consensus sequence contains two conserved regions, an 11-bp
distal region [5'
57 to
47, AAA(a/t)(a/t)T(a/t)TTTT] and a
4-bp proximal region (
44 to
41, AAAA) (13). Mutational analyses indicate that specific positions within the consensus sequence
(
51 to
53 and
41 to
43) are most critical to function (14) and that each region can function alone, with the
proximal region conferring larger effects on the rrnB P1
core promoter (>100-fold) than the distal region (~15-fold
[14]).
The two strongest UP elements, rrnD P1 and rrnB
P1, match the consensus exactly in one of the two regions and contain
some matches in the other. The rrnD P1 UP element
(~90-fold effect) matches the proximal region consensus exactly and
the distal region at 7 of 11 positions (Fig. 5). It lacks the distal
region T-tract, which likely accounts for its three- to fourfold-lower
activity than that of the consensus UP element. The rrnB P1
element contains an exact match to the consensus in the distal region,
but fewer matches in the proximal region; its somewhat smaller
stimulatory effect (33-fold) may reflect the smaller effects on
transcription of the distal region compared to the proximal region.
The UP elements with moderate to low activity (rrnB P2, RNA
II, and merT) contain less extensive similarity to the
consensus. The rrnB P2 upstream sequence contains three of
four A residues found in the proximal consensus (Fig. 5) and also
contains A residues at
45 and
46, an additional feature of some
strong proximal sequences (14). However, it contains only 4 of 11 distal consensus positions and is not protected against DNase I
cleavage upstream of
51. We therefore suggest that its function may
be attributable largely to proximal region interactions. The effect of
the rrnB P2 UP element on lac promoter activity
(~12-fold) was similar to that observed in another study in which
mutant lac promoters containing a series of A residues in
the proximal upstream region conferred an
CTD-dependent increase in
promoter activity (9).
The RNA II and merT UP elements match the consensus better
in the distal than in the proximal region (Fig. 5), which may account in part for their relatively small effects on transcription. We also
note that the RNA II UP element contains two recognition sites for the
Dam methylase (GATC). It has been proposed previously that DNA
methylation plays a role in controlling the RNA II promoter, although
it is not known whether the GATC sites in the UP element, in addition
to a third GATC site in the
35 region, contribute to regulation
(60). The rrnD P1 UP element also contains
a GATC sequence.
The two upstream sequences with negligible effects on transcription
(
pR and lac) have no proximal
region matches to consensus and contain distal regions with either
little similarity to the consensus (lac) or mismatches at
critical positions (
pR). A substitution
mutation in
pR, C to T at
51
(pRM116), that was previously reported to
increase transcription threefold (17) results in a match to
the consensus at eight contiguous positions.
Determinants of UP element strength.
Differences in the degree
of UP element function are likely to reflect several factors,
including (i) the relative affinities of sequences for
subunit,
(ii) the exact positioning of the sites with respect to the other
promoter elements (40, 44), and (iii) the extent to which a
particular core promoter mechanism is rate limited at a step affected
by
subunit-DNA interaction. The affinities of two UP elements
(rrnB P1 and rrnB P2) for purified
subunit
differed by about fourfold (Fig. 4) (51), and this may
account for the difference in their effects on the same core promoter
(hybrid-lac promoters [Table 3]). However, the relative affinities of different sequences for purified
subunit must be
interpreted with caution, since binding of
subunit alone might not
be a reliable indicator of
subunit binding as part of the RNAP
holoenzyme.
The lac and rrnB P1 core promoters responded
similarly to the rrnB P1 UP element, as well as to phased
A-tracts (1, 50). However, differences in the promoter
mechanisms may explain why the rrnB P2 UP element had a
greater effect on the lac core promoter than on its own core
promoter (12.8-fold versus 3.3-fold, respectively [Tables 2 and 3]).
Other core promoter sequences may respond less well to and, in some
cases, may even be inhibited by
CTD-DNA interactions. For example,
an upstream A-tract (presumably an
subunit binding site [see
below]) was reported to increase the activity of one synthetic core
promoter, but to inhibit the activity of a second, clearance-limited
core promoter (12). However, none of the upstream sequences
analyzed in our study had negative effects on promoter activity.
A-tract sequences and
subunit recognition.
Existing data
suggest that the proximal and distal consensus sequences may each
represent an
CTD monomer binding site (14). Each of these
sequences contains an A-tract, and although details of the
CTD-DNA
interaction remain to be defined, the unusual structural features of
A-tract DNA (reviewed in reference 63) may play a
role in its recognition by
subunit. The upstream sequences in our
study that functioned as stronger UP elements (e.g., RNA II,
rrnB P2, rrnB P1, and rrnD P1) contain
an A- or T-tract at least 4 nt in length, consistent with the role of
an A-tract in recognition by
subunit.
We have found that the previously observed stimulatory effects of
multiple phased A-tracts on transcription (24, 49) depend upon interaction with the RNAP
subunit (1). Although
multiple phased A-tracts result in the macroscopic curvature that
confers aberrant gel electrophoretic mobility (35), this
macroscopic curvature does not appear to be essential for UP element
function. A single A-tract in the
40 region can have a large effect
on transcription (14), and some UP elements (e.g.,
rrnB P1) do not display such curvature (20).
Sites of enhanced DNAse I cleavage have been observed in the
footprints of proteins known to bend DNA, such as FIS and CRP (15, 34). The enhanced DNase I cleavage sites in the
upstream regions of several of the promoter-RNAP complexes analyzed
here (Fig. 4 and 5) and in a consensus UP element (13)
suggest that DNA distortion occurs upon
subunit binding. These
upstream hypersensitive sites also indicate that one face of the DNA
helix is accessible to other proteins (DNase I in this case) when
subunit is bound and that
subunit and an activator protein could
interact simultaneously on different surfaces of an upstream sequence,
as suggested for the Ada protein at the ada and
aidB promoters (36).
UP element position and size.
The upstream sequences
characterized in this and previous work were located primarily
between
40 and
60 (Fig. 1 and 5 and Tables 2 and 3).
However, DNA sequences smaller than 20 bp in length can function as
UP elements to increase transcription. For example, the proximal or
distal portions of the rrnB P1 UP element confer partial UP
element function (14, 44, 50). Similarly, some of the UP
elements described in this paper (e.g., rrnB P2 [described
above]) are likely to utilize only a limited portion of the upstream
region for
subunit interactions.
Sequences upstream of
60 may also interact directly with RNAP and
contribute to transcription in some promoters. In the rrnB P1 promoter, sequence upstream of
60 increases activity about 1.5-fold in vivo in the absence of the activator protein FIS (13, 50), and the distal portion of the rrnB P1 UP element
(the
50 region) retains full function and protection in
footprints when moved one turn of the DNA helix upstream of its
normal position (44). RNAP also protects DNA upstream of
60 in some promoters, for example, in the B. subtilis
hag promoter (18). The
CTD can also affect
transcription when positioned several turns upstream of its normal
location in complexes formed with the activator protein CRP (4,
43, 55). This variability in positioning of
subunit binding
sites most likely results from the flexible linker connecting
the
CTD to the
subunit N-terminal domain (5,
32).
The downstream boundary of the UP element region is at around position
40, since sequence between
38 and
40 was not strongly conserved
in the in vitro-selected UP elements (13), and substitutions at these positions had only minor effects on transcription
(14). We note that the identity of the residue directly
adjacent to the
35 hexamer (
37 in our numbering system [see Fig.
5]) is very important to transcription of the rrnB P1,
pR, and lac promoters (2, 10,
33). The effects of this residue on rrnB P1 function
are independent of the
CTD, suggesting that it plays a role in
,
not
subunit, interactions (2).
Modular structure of promoters.
In summary, we conclude that
UP elements occur in a variety of promoters, where they make different
contributions to promoter strength. Thus, promoters can be considered
as modular composites of a series of at least three RNAP recognition
elements: the
10 and
35 hexamers and the UP element. (Our studies
do not exclude the possibility of additional RNAP recognition
determinants as well, e.g., in downstream regions
[7].) Together these RNAP recognition elements confer
appropriate basal activity for a particular promoter in the absence of
transcription factors. Individual promoters need not contain
significant information in all promoter modules, and many promoters
have evolved to utilize transcription factors that respond to specific
environmental signals in lieu of a particular interaction.
We thank Jeremy Fields and Robin Pietropaolo for their assistance
in construction of some of the promoters used in this work and Shawn
Estrem for helpful discussion.
This work was supported by NIH grant GM37048 to R.L.G.
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