(Received for publication, February 25, 1997, and in revised form, April 16, 1997)
From the Department of Molecular Genetics and
Microbiology, University of Massachusetts Medical School, Worcester,
Massachusetts 01655 and the § Department of Bacteriology,
University of Wisconsin, Madison, Wisconsin 53706
Expression of the Escherichia coli
adaptive response genes (ada, aidB, and
alkA) is regulated by the transcriptional activator, Ada.
However, the interactions of RNA polymerase and Ada with these
promoters differ. In this report we characterize the interactions of
Ada, methylated Ada (meAda), and RNA polymerase at the
alkA promoter and contrast these interactions with those
characterized previously for the ada and aidB
promoters. At the alkA promoter, we do not detect the RNA polymerase subunit-mediated binary complex detected at the
ada and aidB promoters. In the presence of
either of these two activators, RNA polymerase protects the
alkA core promoter, including the elements at
35 and
10, and is more efficient in transcription initiation in
vitro. RNA polymerase holoenzyme containing the
subunit
mutation R265A is severely impaired in Ada-independent basal
alkA transcription, shows no activation by Ada or
meAda, and fails to bind the alkA promoter
in vitro. Binding of the purified wild type
subunit to
alkA was not detected, but a complex of promoter DNA, Ada
or meAda, and
was observed in gel shift assays. These
observations suggest that both forms of Ada protein activate
alkA transcription by enhancing RNA polymerase holoenzyme
and
subunit binding.
In bacteria, transcriptional activation plays a pivotal role in
adaptation processes. Changes in the cell's environment stimulate transcriptional activators to bind their target promoters and to induce
their expression. This results in adaptation of gene expression to the
environmental requirements. Transcriptional activators have recently
been classified on the basis of their mechanism of interaction with RNA
polymerase, leading to the definition of Class I and Class II
activators (1). According to this definition, Class I transcriptional
activators work by contacting the subunit of RNA polymerase,
whereas Class II activators interact with the
factor.
and
are also the subunits of RNA polymerase involved in specific DNA
binding and promoter recognition.
is responsible for interaction
with the
35 and
10 sequences, whereas
is able to bind a third
element of the bacterial promoter identified at the rrnB P1
promoter region and termed the UP element (2).
The Escherichia coli adaptive response to DNA methylation
damage is induced when cells are exposed to sublethal doses of
alkylating agents (3-5). ada is the regulatory gene of the
adaptive response. The product of the ada gene, Ada, is a
methyltransferase able to transfer methyl groups from damaged DNA to
two of its own cysteine residues (6, 7). Methylation of Cys-69 converts
Ada into a transcriptional activator. The Cys-69-methylated Ada protein (meAda)1 is able to recognize
specific sequences upstream of its own promoter and at least two other
promoters, alkA and aidB, and activate their
transcription (8-10). meAda has been proposed as a Class I
transcriptional activator, since deletion of the carboxyl-terminal
domain of the RNA polymerase subunit results in the loss of
transcriptional activation by meAda at the ada
promoter (11).
We have recently shown that the subunit of RNA polymerase binds to
the promoters of the adaptive response genes ada and aidB in the absence of Ada protein (12). RNA polymerase
holoenzyme binds the two promoter regions between
60 and
40 via its
subunit; this area overlaps the meAda binding site and
closely resembles the UP element of the rrnB P1 promoter
with respect to location, high A/T content, and recognition by
(2).
This RNA polymerase·promoter binary complex shows only basal levels
of transcription and is modified by meAda into a ternary
complex competent in transcription initiation at induced levels (12).
The single amino acid substitution in the
DNA binding domain,
R265A, which is a change from arginine to alanine at residue 265, prevents
from binding the rrnB P1 promoter (13) as well
as the
60 to
40 region of the ada and aidB
promoters (12).
The alkA gene encodes a glycosylase responsible for
recognition and excision of methylated DNA bases (14). A number of
observations strongly suggest that the mechanism of Ada action on the
alkA promoter differs from that of the other adaptive
response genes. First, methylation of Ada is not necessary for
alkA induction. Overexpression of the unmethylated Ada
in vivo is sufficient to induce alkA
(i.e. in the absence of alkylation damage) (14). Both Ada
and meAda proteins are able to activate transcription of
alkA in vitro (9), although higher concentrations of Ada are
required (15, 16). In contrast, the ada and aidB
promoters are only activated by meAda protein (6, 8), and
high concentrations of Ada protein inhibit activation by
meAda (17). Second, the location of the Ada binding site in
the alkA promoter region differs from that in the
aidB and ada promoters. At the alkA
promoter, meAda protects residues from 54 to
29 (16,
18), overlapping the
35 region. In both ada and
aidB, meAda binds farther upstream, protecting
sequences from
62 to
38 in DNase I protection experiments (10, 19)
(see Fig. 1). Third, Ada mutants capable of activating alkA
but not ada, and vice versa, have been isolated (15, 20).
Likewise, the isolated, methylated amino-terminal domain of Ada acts
differently on the two types of promoters, activating alkA
but inhibiting ada (18).
In this report we investigate the interaction between RNA polymerase
and the alkA promoter and the effects of Ada and
meAda on this interaction. We find that 1) the RNA
polymerase subunit plays an important role in both basal and
Ada-activated transcription, 2) both Ada and meAda
stimulate specific binding of both RNA polymerase holoenzyme and the
purified
subunit of RNA polymerase, and 3) the Ada·RNA polymerase·alkA promoter ternary complex differs from the
ternary complex formed by meAda in the extent of upstream
DNA binding.
Restriction endonucleases were from Boehringer
Mannheim. [-32P]dATP and [
-32P]CTP
were from Amersham Life Science. E. coli RNA polymerase and
RNase-free DNase I were from Pharmacia Biotech Inc.
An
NruI/PstI (60 to +296 of the alkA
region) fragment from pYN3072 (9) was subcloned into pSL1180
(Pharmacia) using the corresponding restriction sites to obtain plasmid
pMV447. To obtain pMV465, pMV447 was cut with AccI, and the
vector fragment was re-ligated to delete 253 bp from the polylinker of
pSL1180. For the coding strand, a HindIII/AccI
(145 bp) fragment from pMV447 that contained the alkA
promoter region from
60 to +24 plus an additional 61 bp of upstream
vector DNA was labeled at the AccI site with
[
-32P]dATP by an end-filling reaction with Klenow
enzyme. For the template strand, a HindIII/EcoRI
(169 bp) fragment from pMV465 that contained the same 145-bp fragment
as pMV447 plus an additional 24 bp of downstream vector DNA was labeled
at the HindIII site. The region downstream of
60 contained
all the necessary elements for transcription and regulation of
alkA (Ref. 16 and Fig. 1). Deletion of the
region upstream of
60 resulted in the loss of a second promoter that
reads in the opposite direction (14). This promoter is not regulated by
the Ada protein and does not affect levels of alkA
transcription either in vivo or in
vitro.2 The Ada protein was purified
as in Saget and Walker (17), and methylation of the Ada protein was
obtained as in Nakabeppu and Sekiguchi (9). DNase I protection and gel
retardation experiments were performed in 20 µl final volume of
binding buffer (12). Samples were incubated for 20 min at 22 °C and
processed as described previously (12).
Single-round in vitro
transcription experiments were performed using the linear DNA templates
as follows. For the alkA promoter, a
HindIII/EcoRI fragment from pMV465 (169 bp) was
used. This fragment contained the same 169-bp alkA promoter
fragment used in DNase I protection experiments of the template strand.
The RNA transcript obtained from this fragment was 48 nucleotides in
length. As a control, we used the lacUV5 promoter, a 205-bp
fragment from pYN3077 (9), to produce an RNA transcript of 65 nucleotides. In the experimental conditions, a second RNA transcript
approximately 10 nucleotides shorter was also produced, possibly from a
cryptic promoter on the same DNA fragment. The sizes of the RNA
transcripts were confirmed by DNA sequencing ladders run on the same
gels, assuming an electrophoretic mobility 10% slower for RNA than
DNA. 0.3 pmol of each promoter region were preincubated for 5 min at room temperature with 4 pmol of either wild type RNA polymerase holoenzyme or R265A-RNA polymerase (reconstituted as in Ref. 13) in
40 mM Tris-HCl, pH 8.0, 30 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol, and
20 µg/ml bovine serum albumin. When required, 10 pmol of unmethylated
or methylated Ada were added, and the mixture was incubated for 15 min
at 37 °C. Methylation of Ada was performed as in Ref. 9, except that
the amount of methylated calf thymus DNA was lowered to 0.06 µg.
Transcription reactions were started by the addition of 0.1 mM each ATP, GTP, and UTP, 0.001 mM CTP, 10 µCi of [
-32P]CTP, 250 µg/ml heparin (final
reaction volume, 50 µl). After 5 min of incubation at 37 °C, 1 mM unlabeled CTP was added, and the reaction was allowed to
continue for 3 min and then blocked with 50 µl of stop solution (600 mM NaCl, 50 mM EDTA, pH 8.0, 30 µg/ml salmon
sperm DNA). Samples were precipitated with 2 volumes of ethanol,
resuspended in gel loading buffer, and analyzed on a 6%
polyacrylamide, 8 M urea gel.
Consistent with previous reports (8, 9, 16), we
observed a low level of transcription of the alkA promoter
with wild type RNA polymerase in vitro in the absence of any
activator protein (Fig. 2, lane 2). Both the
unmethylated and the methylated forms of Ada (Ada and
meAda) stimulated in vitro transcription,
although the highest levels of transcription occurred with
meAda (Fig. 2, lanes 3 and 4). We and
others have previously found that the carboxyl-terminal domain of the
RNA polymerase subunit plays a role at other Ada-activated
promoters (11, 12). However, the binding site for meAda in
the alkA promoter (
52 to
30, partially overlapping the
35 region) differs from that at the aidB and
ada promoters that have meAda binding sites
extending from
36 to
60 and
40 to
64, respectively. This result
combined with the results from the genetic and biochemical studies of
others (11, 17, 18, 20) suggests that the mechanisms at these promoters
may differ. We therefore asked whether the carboxyl-terminal domain of
the RNA polymerase
subunit is essential for activation of
transcription by Ada at the alkA promoter.
We used an RNA polymerase containing the R265A mutation. This
mutant
subunit was previously shown to be defective in DNA binding
and, when assembled into the RNA polymerase holoenzyme, was defective
in UP element function at the rrnB P1 promoter (13). Purified
R265A was also shown to be defective in the binding of the
upstream regions of the Ada-dependent aidB and
ada promoters (12). The
R265A-RNA polymerase was
reconstituted in vitro and used in transcription of
alkA in the presence or absence of Ada and meAda
(Fig. 2, lanes 5-8). The activator-independent, basal level of transcription of alkA was reduced substantially by the
R265A mutation (lane 6), and no activation of trancription
was detected by either Ada or meAda (lanes 7 and
8). The R265A-RNA polymerase transcribed the lacUV5 promoter as efficiently as wild type RNA polymerase
in these reactions (compare lanes 1-4 with lanes
5-8). Another transcript from the placUV5 fragment was
also detected with wild type RNA polymerase (lanes 1-4)
although not with R265A-RNA polymerase (lanes 5-8). This
transcript is approximately 52 nucleotides in length and may be the
result of either initiation from a weak promoter-like sequence or
premature termination.
Since unmethylated Ada was found to stimulate
transcription by wild type RNA polymerase, but less effectively than
meAda (Fig. 2 and Ref. 9), we tested the possibility that
there are differences in the affinity of the two forms of the activator for the binding site in the alkA promoter. We used a gel
shift assay to compare the concentrations of Ada or meAda
required to observe formation of a complex with an alkA
promoter fragment (Fig. 3). A meAda·DNA
complex was formed that was detectable at a relatively low
meAda concentration (0.015 µM) and required a
concentration of approximately 0.05 µM for complete site
occupancy. Complexes of higher mobility were also formed at high
concentrations of meAda, but the nature of the interactions
in these complexes is not understood. In contrast, the unmethylated
form of Ada formed complexes with alkA only at a relatively
high concentration (0.17-0.50 µM), and full site
occupancy was not detected at the maximum concentration used. Thus, the
methylated form of Ada binds the alkA site more efficiently.
Formation of Complexes of alkA, RNA Polymerase, and Either Ada or meAda
We previously observed that RNA polymerase
alone, in the absence of meAda, protected the upstream
region of the aidB and ada promoters in a complex
detected by DNase I footprinting. The basal level of alkA
transcription by the wild type RNA polymerase (Fig. 2) indicates that
RNA polymerase is also able to specifically recognize the
alkA promoter in the absence of activator. However, we were unable to detect this RNA polymerase·alkA complex in a
DNase I footprint (Fig. 4, compare lane 1 with 4), presumably reflecting the instability of the
complex under these conditions. Consistent with the reduced ability of
the R265A mutant RNA polymerase to carry out basal transcription of
alkA (Fig. 2), we were also unable to detect a complex of
this mutant RNA polymerase with the promoter (Fig. 4, lane
7).
A stable ternary complex of alkA promoter, RNA polymerase,
and either Ada (Fig. 4, lane 5) or meAda (Fig.
4, lane 6) was observed. The boundaries of protection conferred by the activator protein alone (52 to
30) are seen in the
footprint of meAda (Fig. 4, lane 3), consistent
with previous results (18). Although the unmethylated form of Ada does
not bind well enough to confer clear protection in the DNase I
footprints (Fig. 4, lane 2; see also Figs. 3 and 5), it does
support the formation of a ternary complex with RNA polymerase and the
promoter. The upstream boundaries of protection observed with Ada and
meAda containing ternary complexes differ (compare
lane 5 with 6). The complex containing
meAda had an upstream boundary of
52, whereas the complex
with Ada had a boundary at
43. Increasing the concentration of Ada to
2.5 µM did not convert the protection pattern exhibited
by Ada to that of meAda (data not shown), suggesting that
the difference in protection patterns reflects differences in the
structure of the complexes, rather than degree of occupancy of the
promoter. The Ada or meAda concentration dependence of
formation of these complexes is examined further in Fig.
5.
Consistent with the inability of the mutant R265A-RNA polymerase to be
activated by Ada or meAda at the alkA promoter
(Fig. 2), we did not observe a ternary complex of R265A-RNA polymerase,
the alkA promoter, and Ada or meAda in DNase I
footprint assays (Fig. 4, lanes 8 and 9). (The protected region in lane 9 reflects binding of
meAda (compare with lane 3).) Thus, the arginine
265 residue in the carboxyl-terminal domain of the subunit is
required for formation of the Ada- or meAda-stimulated
ternary complexes.
Consistent with the gel shift results of Fig. 3 indicating that only a small fraction of DNA molecules were bound by Ada, we did not detect an Ada footprint at concentrations ranging from 0.02 to 0.5 µM (Fig. 5, lanes 2-4). Over a comparable concentration range, meAda fully occupied its alkA promoter binding site (Fig. 5, lanes 5-7). Although binding of unmethylated Ada protein alone was not detectable, it stimulated the formation of a ternary RNA polymerase·alkA·Ada complex at each concentration tested (Fig. 5, lanes 9-11), as did meAda (lanes 12-14). Thus, it appears that there is cooperative interaction of RNA polymerase and Ada at this promoter.
Formation of Complexes ofThe carboxyl-terminal
domain of the RNA polymerase subunit plays a role in both basal and
activated transcription at alkA (Fig. 2). The effect on
basal transcription indicates that the alkA promoter
contains a UP element component. Purified
subunit binds to DNA in
the UP element region of several promoters, including the
meAda-activated ada and aidB
promoters (12). However, we were unable to detect complexes of purified
subunits and the alkA promoter in a gel shift assay at
concentrations higher than required for complex formation at other UP
elements (0.5 µM (13); Fig. 6, lane
4) or in DNase I protection assays (data not shown). In the
presence of purified wild type
subunit and either Ada or meAda, complexes with mobility slower than that of the Ada
or meAda binary complexes were observed, indicating a
cooperative interaction of
with both forms of Ada (Fig. 6, compare
lanes 5 and 6 with lanes 2 and
3). The
·Ada or
·meAda complexes were
not detected, however, when purified R265A mutant
subunit was used
(lanes 7-9), indicating that the DNA binding function of
the carboxyl-terminal domain is necessary for the formation of these
complexes as well as for activation of this promoter.
In a previous report (12), we showed that RNA polymerase is able
to contact the promoters of the adaptive response genes ada
and aidB via binding of the subunit to the
40 to
60
region in the absence of the transcriptional activator
meAda. At these promoters, binding of meAda
converts the RNA polymerase·promoter complex into a ternary complex
competent for induced levels of transcription initiation. At the
ada and aidB promoters, only meAda
functions as an inducer, and its role in activation is to isomerize a
binary complex of RNA polymerase bound to an upstream site via
subunit contacts; binding of meAda alters the nature but
not the affinity of RNA polymerase binding, leading to complete
promoter protection and induced transcription (12). The alkA
promoter differs from ada and aidB promoters in
several respects. Both forms of the Ada protein activate
alkA transcription (9) and stimulate complete protection of
the alkA promoter region by RNA polymerase (Fig. 4,
lanes 5 and 6). RNA polymerase alone binds the
alkA promoter too weakly to afford clear DNase I protection,
but both forms of Ada activator enhance RNA polymerase binding to the
alkA promoter. Thus, the role for meAda at the
alkA promoter is fundamentally different from its role at
other Ada-dependent promoters.
Although RNA polymerase is unable to bind the alkA promoter in the absence of Ada at levels sufficient to afford clear protection from DNase I (Fig. 4, lane 4), it can specifically interact with the alkA promoter region, resulting in low levels of transcription in vitro (Fig. 2, lane 2) and providing a basal level of expression of alkA in vivo (21).
The region of alkA bound by meAda extends from
52 to
30 (Ref. 18 and Fig. 4, lane 3). The weak binding
displayed by unmethylated Ada in the absence of RNA polymerase does not
allow a determination of its binding site. The
52 to
30 area
includes the recognition sequence we have recently proposed for
methylated Ada, AATnnnnnnGCAA (where n represents the part of the
sequence that varies) (10), which in the alkA promoter spans
between
46 and
34 (Fig. 1). The binding affinity of
unmethylated Ada to the alkA promoter region in the ternary
complex is increased at least 25-fold (Fig. 5) by the presence of RNA
polymerase. Unlike the unmethylated protein, meAda binding
affinity to the alkA promoter region is not affected by RNA
polymerase in this concentration range (Fig. 5).
Differences between Ada· and meAda·RNA
polymerase·alkA promoter ternary complexes are clearly
detectable in DNase I protection experiments in the 40 to
60 areas
(Fig. 4). These differences appear to be qualitative rather than
quantitative, since different DNase I protection patterns are produced
even in the presence of a large excess of unmethylated Ada (2.5 µM, data not shown). We suggest that different DNase I
protection patterns reflect different conformations of RNA polymerase
and/or promoter DNA in the two ternary complexes. Despite these
differences, both unmethylated and methylated Ada share the ability to
stimulate
binding and trigger more efficient transcription
initiation.
In contrast to what is seen at the ada and aidB
promoters, the subunit of RNA polymerase does not bind the
alkA promoter in the absence of the Ada protein; however,
the presence of either Ada or meAda together with
results in formation of ternary complexes detectable in band shift
experiments (Fig. 6, lanes 5 and 6). The
requirement for the binding of Ada protein to the alkA
promoter for
binding raises the possibility that alkA
promoter activation may result from recruitment of
to its binding
site, which in turn allows RNA polymerase to bind the appropriate core
promoter elements. Ada- and meAda-stimulated binding of
could be mediated by protein-protein interaction of
with Ada and
meAda; an alternative possibility is that binding of Ada or
meAda might facilitate binding of
to a specific DNA
site in the alkA promoter. The observation that the DNA
binding-defective R265A mutant
subunit is not able to bind the
alkA promoter region even in the presence of
meAda is consistent with this hypothesis. Promoters have
already been described in which the
subunit is able to specifically bind DNA in the
40 to
60 area upon binding of transcriptional activators: in the gal P1 promoter, for instance, binding of
the catabolite repressor protein induces upstream binding of
(22). DNase I protection assays performed on the
meAda·
·alkA promoter ternary complex
indicate that
does not extend the area of protection provided by
meAda (data not shown), suggesting that the
binding
site overlaps the meAda binding site and is therefore
indistinguishable in DNase I protection experiments. Overlapping sites
for
and meAda have already been observed at both the
ada and aidB promoters (12).
The R265A mutation of impairs RNA polymerase binding to the
alkA promoter in the presence of Ada, as determined by DNase I protection experiments (Fig. 4) and almost completely abolishes basal
and activated transcription from the alkA promoter (Fig. 2).
These observations suggest that the
subunit plays an important role
in the formation of the RNA polymerase·alkA promoter
binary complex and that
binding is necessary for uninduced, basal
levels of alkA transcription. This finding is apparently a
contradiction with the lack of binding observed with the purified
subunit (Fig. 6). However, additional interactions between the promoter and the other subunits of RNA polymerase may enhance or stabilize
binding when it is part of the holoenzyme. Indeed, differences in
concentration needed for DNA binding by the
subunit alone and
assembled into RNA polymerase have already been described (2, 12). At
the alkA promoter, the binding of RNA polymerase subunits in
other areas of the promoter might provide stabilization of
binding.
Moreover, the requirement of Ada or meAda for
subunit
binding suggests that the different role for Ada in activation of
alkA predicted from genetic studies (9, 14-17) may be that
at alkA, Ada and meAda function to enhance RNA
polymerase holoenzyme binding by stimulating
binding, whereas at
the ada and aidB promoters, only
meAda functions as an activator, and
binding is
unaffected by meAda; it has been proposed that at
ada and aidB, meAda is required for
binding of RNA polymerase to the core promoter elements at
10 and
35 (12).
Although the DNA binding function of the subunit is required for
binding of RNA polymerase to the alkA promoter, this
requirement does not exclude the possibility that transcription is
activated by Ada through a Class I transcriptional activator mechanism, as proposed (1, 11). At the alkA promoter, methylated Ada binding is required for
binding, a role consistent with the general
model proposed for Class I transcription activators (23). It is
possible that interaction of the Ada protein with the
subunit
triggers an extensive conformational change in RNA polymerase, so that
the
35 and
10 elements are accessible to the
factor, and
transcription initiation can occur. However, direct interaction of Ada
and meAda with other subunits such as
cannot be ruled
out; the location of the Ada binding site in the alkA
promoter would favor such interaction, which might not be possible in
the other Ada-dependent promoters where the Ada binding
site is 6-8 base pairs upstream of the
35 sequence.
We thank Richard L. Gourse for critical reading of the manuscript.