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INTRODUCTION |
Transcription activation is one of the principal strategies used
by bacteria to respond to external stimuli and to adapt to a changing
environment. Most Escherichia coli activators stimulate transcription by establishing protein-protein interaction with RNA
polymerase. Different subunits of RNA polymerase can be a target for
transcription activators; however, the majority of activators interact
with either the
or the
70 subunits (1, 2).
and
70 are also the subunits of RNA polymerase responsible
for specific binding to promoters;
70 contacts the
35
and
10 promoter elements (core promoter elements), whereas
interacts with UP elements. At the strong rrnB P1 promoter, an UP element stimulates transcription initiation 30-fold through direct interaction with the
subunit C-terminal domain
(
CTD),1 in the absence of
any other protein factors (3).
Exposure of E. coli to sublethal concentrations of
methylating agents such as methyl methanesulfonate (MMS) activates
expression from three promoters: the ada promoter (which
also controls expression of the alkB gene), the
alkA promoter, and the aidB promoter. This process is called the adaptive response (4-7). The product of the
ada gene, the Ada protein, plays a dual role in the adaptive response; Ada transfers methyl groups from DNA to two of its cysteine residues, thereby functioning as a DNA repair protein. Upon
self-methylation, Ada is converted into an activator able to stimulate
transcription of the adaptive response genes, including its own
(8-10). Ada is a 39-kDa protein organized in two independently
structured domains, each with one methyl-acceptor cysteine (11).
Methylation of Cys-69, in the N-terminal domain, triggers specific DNA
binding by Ada and is required for transcription activation. In
contrast, Ada protein singly methylated at Cys-321 is not capable of
specific DNA binding activity. However, the Ada C-terminal domain does play a role in transcription activation; deletions in Ada CTD affect
transcription of ada, even though they do not affect
specific DNA binding (12-14). The methylated Ada protein
(meAda) recognizes the sequence AAT(N)6GCAA,
which at ada and aidB is located 5 and 7 base
pairs upstream of the
35 sequence, respectively (10, 15).
In the model for meAda activation previously proposed (16),
meAda contacts RNA polymerase through protein-protein
interaction with
CTD and recruits RNA polymerase to the promoter
region (15, 16). However, in a previous report (17), it was shown that RNA polymerase binds the ada and aidB promoters
via
CTD, regardless of the presence of Ada. In the absence of
meAda, RNA polymerase binds to the
60 to
40 region,
which also includes the meAda binding site. This region
closely resembles the UP element of the rrnB P1 promoter in
A/T content, in location, and in its function as a binding site for the
subunit of RNA polymerase. Mutations in
CTD that abolish
binding to the rrnB P1 UP element also affect binding to the
60 to
40 region of ada and aidB (17) as well
as transcription activation by meAda (16, 18). Due to these
similarities, the
60 to
40 regions of ada and
aidB can be considered as "UP-like elements." UP-like elements promote RNA polymerase binding to the ada and
aidB promoters in the absence of meAda, but the
resulting RNA polymerase-promoter binary complexes can only initiate
transcription with poor efficiency. Binding of meAda
promotes the formation of a ternary complex that is proficient in
transcription initiation (17).
In this report, we show that although
CTD is responsible for the
formation of the initial RNA polymerase-promoter binary complex,
transcription activation requires protein-protein interaction between
meAda and the C-terminal domain of the
70
factor of RNA polymerase (
70 CTD). Our observations
suggest that
and
70 CTD are necessary for different
steps in transcription initiation at the ada and
aidB promoters.
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EXPERIMENTAL PROCEDURES |
Construction of aidB Hybrid Promoters and Promoter Activity in
Vivo--
The minimal sequence requirement for
Ada-dependent activation of the aidB promoter is
shown in Fig. 1. This fragment was subcloned into the multiple cloning
site of the plasmid pSL1180 (Amersham Pharmacia Biotech), producing
plasmid pPL115. The substitutions of the
10 and
35 promoter
elements to consensus sequences, and of the aidB
60 to
40 region to the rrnB P1 UP element (see "Results"), were introduced by using double strand oligodeoxynucleotides. Plasmid pPL115 was digested with either BamHI and
NcoI or NcoI and MluI and religated in
the presence of complementary double strand oligodeoxynucleotides
carrying the desired substitutions and the corresponding restriction
sites. The ligation mixtures were used to transform E. coli
strain RB791 (19), and recombinant plasmids were sequenced using the T7
sequencing kit (Pharmacia). The promoters were then tested for their
in vivo activity by
-galactosidase assays in the
rpoS strain MV2792. Deletion of the rpoS gene
completely abolishes ada-independent regulation of
aidB (20, 21). Strains were grown to 0.2 A600 nm in LB medium supplemented with 0.2%
glucose, 20 µg/ml tetracycline, and 80 µg/ml ampicillin and then
rediluted 1:50. At an A600 nm of about 0.02, the cultures were divided in two aliquots, and one was supplemented
with 0.04% MMS to activate the adaptive response. Samples were taken
2 h after induction, and
-galactosidase activity was measured
as described in Ref. 22. For
-galactosidase experiments with wild type and mutant rpoD alleles, strain MV3766
(alkB::
PSG1 camR
lacZ) was used.
-Galactosidase activity was measured as
described above, except that 25 µg/ml chloramphenicol was added to
the medium, and MMS induction was started at 0.1 A600 nm.
In Vitro Transcription--
Reconstitution of RNA polymerase
with histidine-tagged full-length
or histidine-tagged truncated
-235 was performed as in Ref. 23. No contamination from wild type
was detectable by SDS-polyacrylamide gel electrophoresis in the
-235 RNA polymerase preparation. For reconstitution of RNA
polymerase with wild type
factor or the E575V mutant,
histidine-tagged
was purified using Ni-NTA columns (Quiagen), using
the standard protocol provided by the manufacturer. Purified
factors were added at a 4:1 ratio to core RNA polymerase (Epicentre).
Ada was purified as in Ref. 14 and methylated prior to use by the
method reported in Ref. 9; when necessary, 0.2 µM of
meAda was added to the transcription reaction mixture.
Single-round in vitro transcription experiments from linear
templates were performed as in Ref. 18. 5 pmol (0.1 µM)
of reconstituted RNA polymerases was used, except for
-235 RNA
polymerase, when 12.5 pmol (0.25 µM) was used. For the
experiments in Fig. 3, the DNA templates were
EcoRI-ScaI fragments from pPL115 (wild type
aidB promoter sequence) or from pPL116 (aidB
derivative with a perfect consensus
10 sequence). Both fragments are
166 base pairs long and produce an RNA transcript of 40 nucleotides. A
DNA fragment carrying the lacUV5 promoter was used as
internal control. The fragment is 205 base pairs long and produces a
transcript of 65 nucleotides (9). For the experiments in Fig. 8, the
template was a HindIII-EcoRI fragment from
pYN3066 for the ada promoter (9) and an
EcoRI-ScaI fragment from pPL115, described above, for aidB. The amount of transcription was quantified after
normalization to the lacUV5 transcript using a phosphoimager
(Molecular Dynamics).
Gel Retardation Assays--
Fragments for gel retardation assays
were the same as those used for the in vitro transcription
experiments. Fragments were labeled, and 5,000 cpm/sample was used in
20 µl final volume of reaction buffer (50 mM Tris-HCl, pH
7.6, 50 mM NaCl, 2.5 mM dithiothreitol, 6.25%
glycerol, 10 µg/ml herring sperm DNA). Wild type and mutant RNA
polymerases,
factors (purified as in Ref. 24), and
meAda were added as described in the figure legends.
Samples were incubated 20 min at 37 °C and loaded either on to a 4%
(for experiments with RNA polymerase) or a 6% (for experiments with
factors) native polyacrylamide gel. Gels were run at 10 V/cm in
0.25 × TBE (22.5 mM Tris borate, 0.5 mM
EDTA), 1.25% glycerol. Bands were visualized by autoradiography.
Screening of rpoD Mutations--
To screen for rpoD mutant
alleles affecting ada-dependent transcription, a
plasmid library carrying mutations resulting in single alanine
substitutions at 17 amino acids of
70 C-terminal domain
(obtained from C. Gross, University of California, San Francisco;
plasmids are derivatives of pGEX-2T
70 (25)) was used to
transform MV3766
(ada-alkB::lacZ). Strain MV3766 carries a lacZ transcriptional fusion in the
alkB gene, which lies downstream of the ada gene
and whose transcription is driven by the ada promoter.
Additional mutagenesis of the terminal segment of the rpoD
gene was performed by two independent polymerase chain reactions from
an XhoI site (corresponding to amino acid 528 of
70) to a HindIII site immediately downstream
of the stop codon. Strain MV3766 was transformed with the plasmid
library. Transformants were plated on LB medium and replica-plated onto
MacConkey medium both in the presence and in the absence of 0.02% MMS.
In the presence of MMS, colonies with normal levels of
ada-dependent transcription are pink, whereas
colonies with decreased levels are white. The replica-plating step is
necessary to avoid exposure of the colonies to the mutagenic effects of
MMS. Colonies corresponding to the mutant candidates were picked from
the MacConkey plates with no MMS and tested for
-galactosidase
activity as described above.
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RESULTS |
In Vivo Transcription from aidB Promoter Derivatives--
Fig.
1 shows the sequence of the
ada and aidB promoters. The
60 to
40 regions
of the two promoters show a high degree of similarity to the UP element
of the rrnB P1 promoter. This region is also the binding
site for meAda, and its deletion abolishes
ada-dependent activation (Ref. 26; Fig.
2). Although both promoters have
recognizable
35 and
10 sequences, they differ from the consensus
hexamers TTGACA and TATAAT. At positively controlled promoters, one of
the functions of activator proteins is to improve recognition of one or
more weak promoter elements by RNA polymerase through either
protein-protein interaction or modification of local DNA structure. To
understand which of the weak promoter elements is the target of
meAda activation, we constructed a set of derivatives of
the aidB promoter in which either the
35 or the
10
elements were substituted by perfect consensus hexamers, and the
UP-like element was substituted by the UP element of rrnB
P1. We investigated the effects of these substitutions on both basal
and ada-activated levels of transcription in
vivo.

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Fig. 1.
Sequence of the ada and
aidB promoters. Numbering is relative to the
transcription start. The 35 and 10 elements are shown in
boldface type. The -binding sites (UP-like elements) are
underlined. The location of the restriction sites used for
the introduction of the mutagenic oligonucleotides in aidB
are italicized.
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Fig. 2.
In vivo transcription of aidB
promoter derivatives (performed as in "Experimental Procedures").
Open bars, -galactosidase levels in the absence of MMS.
Filled bars, -galactosidase levels in cells treated with
0.04% MMS. Data are the average of four independent experiments. The
standard deviation was <15%.
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Results shown in Fig. 2 suggest that at the rrnB UP
element/aidB hybrid promoter (aidB
"rrnB UP"), ada-independent transcription levels are only slightly higher than for the wild type aidB
promoter (1.6-fold, Fig. 2). Thus, meAda does not activate
transcription by converting the
binding site of aidB
into a better UP element. Because this substitution also replaces the
Ada binding site, ada-dependent activation is almost completely abolished. Changes to consensus
35 or
10
sequences did have more pronounced effects on transcription; the
consensus
10 increased ada-independent transcription
levels by almost 5-fold, and introduction of a consensus
35 resulted
in a more than 12-fold increase. However, neither substitution
completely relieved the dependence on meAda for optimal
promoter expression; meAda activates the aidB
promoter derivative with a consensus
10 element (aidB
"
10 con") by 5-fold, and the derivative with a consensus
35
element (aidB "
35 con") by 2.3-fold (Fig. 2). These observations suggest that meAda activates transcription by
affecting RNA polymerase interaction with both the
35 and
10
elements of aidB.
In Vitro Transcription--
Sakumi et al. (16) had
proposed that meAda activates transcription by direct
contact with the CTD of the
subunit. We further investigated the
role of
CTD in activation by meAda by performing
in vitro transcription experiments using two forms of
reconstituted RNA polymerase different with respect to their
subunits: one form carried wild type
, the other a mutant
deleted of the C-terminal 94 amino acids (
-235). Although
-235 RNA polymerase is impaired in UP element utilization and in
transcription from some activator-dependent promoters, it
is proficient in transcription from factor-independent and promoters
dependent on activators that do not interact with
(1).
We tested the two forms of reconstituted RNA polymerase for
transcription from the wild type aidB promoter
(aidB wt) as well as from an otherwise identical promoter in
which the
10 sequence was changed to consensus (aidB
"
10 con"). The latter promoter shows 5-fold higher basal
transcription level in vivo compared with the wild type
aidB promoter but is still dependent on meAda
for maximal promoter expression (Fig. 2). Deletion of the
CTD
dramatically affects both meAda-dependent and
independent transcription from the wild type aidB promoter,
even though activation by meAda is not totally abolished
(Fig. 3, lanes 5 and
6). Transcription from aidB "
10 con" by
-235 RNA polymerase is also affected; however, at this promoter
meAda activates transcription by both forms of RNA
polymerase to a similar extent (5.2-fold for wild type
- and 4-fold
for
-235 RNA polymerase; Fig. 3, lanes 7-10). These
results strongly suggest that, although
CTD is necessary for
efficient transcription at the wild type aidB promoter, it
is not required for activation by meAda.

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Fig. 3.
In vitro transcription with reconstituted
wild type (0.1 µM) and -235 (0.25 µM)
RNA polymerases. The filled arrow head shows the
position of the transcript from the lacUV5 promoter
(placUV5). The open arrow head indicates the
position of transcripts from either the wild type aidB
promoter (paidB wt, lanes 3-6) or the
aidB " 10 con" promoter (paidB 10 con,
lanes 7-10). Fold-activation by meAda is shown
below the transcripts.
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The results of in vitro transcription experiments raise the
possibility that
-235 RNA polymerase is not proficient in carrying out transcription from the wild type aidB promoter because
it is affected in RNA polymerase-promoter interaction, rather than interaction with meAda. To investigate this possibility, we
tested both wild type and
-235 RNA polymerase for their ability to
bind the wild type aidB promoter in the absence of
meAda. As shown in Fig. 4,
wild type RNA polymerase binds aidB wt at 0.04-0.08
µM, whereas
-235 RNA polymerase fails to bind the promoter at concentrations up to 0.32 µM. A similar
result was obtained for the wild type ada promoter (data not
shown). Both forms of RNA polymerase were equally as efficient in
binding both the lacUV5 and the galP1 promoters
under the same experimental conditions (data not shown). The above
results show that
CTD promotes recruitment of RNA polymerase to the
ada and aidB promoters independently of
meAda.

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Fig. 4.
Gel retardation experiments performed with
wild type and -235 forms of RNA polymerase. Lanes 1 and
6, wild type aidB promoter, no proteins added.
Lanes 2-5, increasing concentrations of wild type RNA
polymerase (0.04, 0.08, 0.16, 0.32 µM). Lanes
7-10, same concentrations of -235 RNA polymerase. F
indicates the free DNA fragment.
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Gel Retardation Studies with meAda and
70--
The location of meAda binding sites
at the ada and aidB promoters is consistent with
the possibility of interaction with the
70 factor of RNA
polymerase. To investigate this possibility, gel retardation
experiments were performed with meAda and purified
70.
70 is capable of specific DNA binding
only when assembled into RNA polymerase holoenzyme (27). Indeed, no
binding of
70 alone to either the ada or
aidB promoters could be detected; however, addition of
70 (0.5 µM) to a
meAda·promoter complex resulted in a supershift at both
the ada (Fig. 5) and the
aidB (data not shown) promoters. This suggests that
70 is able to form a ternary complex with
meAda at either the ada or the aidB
promoter DNA. The presence of multiple shifts is possibly due to
partial dissociation of the meAda·DNA complex in the
experimental conditions used and is a typical pattern for
meAda (10). In control gel retardation experiments, no
significant binding by meAda and
70 was
detected with unrelated DNA fragments;
70 was unable to
promote binding of the unmethylated Ada protein to the ada
promoter. Finally,
70 did not produce any supershift
when added to a CRP·gal P1 promoter complex (data not
shown).

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Fig. 5.
Gel retardation experiments with purified
70 factor at the ada promoter
(Pada). Lane 1, no proteins added; lane
2, 70 (0.5 µM); lane 3,
meAda (0.4 µM); lane 4,
70+meAda. F indicates the free
DNA fragment; CI indicates the meAda·DNA
complexes; CII indicates the
70/meAda·DNA complexes.
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Gel retardation experiments were performed with two
70
deletion mutants,
574 and
529; these
mutants, deleted of 39 and 84 amino acids in the C-terminal domain,
respectively, are impaired in interaction with some activators when
assembled into RNA polymerase holoenzyme (24). Concentrations up to 5 µM of the two mutant
factors promote little formation
of ternary complexes at either the ada (Fig.
6) or the aidB (data not
shown) promoter; in the same experiment, complete supershift of the two
complexes occurred at 1 µM with wild type
70.

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Fig. 6.
Gel retardation in the presence of
meAda and wild type and truncated factors at the
ada promoter (Pada). When present,
concentration of meAda was 0.4 µM.
F indicates the free DNA fragment; CI indicates
the meAda·DNA complexes; CII indicates the
70/meAda·DNA complexes.
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Substitutions in
70 Affect
meAda-dependent Transcription--
The above
gel retardation experiments suggest that the C-terminal domain of
70 could be the target for activation by
meAda. Therefore, we expected mutations in the terminal
segment of the rpoD gene to specifically affect
ada-dependent transcription. We screened for
this class of rpoD mutants using a plasmid library carrying
rpoD alleles in which the segment encoding for amino acids
528-613 of
70 had been mutagenized by polymerase chain
reaction. The plasmid library was used to transform strain MV3766. This
strain carries a lacZ transcriptional fusion in the
chromosomal alkB gene, whose transcription is driven by the
ada promoter. Over 1700 colonies were screened and four
mutant colonies were isolated. The mutant rpoD alleles were
sequenced; three carried nonsense mutations, which resulted in
truncations of
70 at amino acid 560 (1 clone) or at
amino acid 584 (2 clones). The fourth candidate carried a missense
mutation, resulting in a glutamic acid to valine change at position 575 of
70 (E575V).
In addition to polymerase chain reaction-directed mutagenesis, we also
tested a set of plasmids (kindly given by C. Gross, UCSF) carrying
rpoD alleles with single alanine substitutions at 17 amino
acids of
70CTD (Fig. 7).
-Galactosidase assays were performed to quantify the effect of the
alanine substitutions, as well as the E575V mutation, on transcription
from the ada promoter. In strain MV3766, wild type
70 from the chromosomal rpoD gene is normally
expressed, and mutated
factors are present in only a slight excess
to wild type
70 (data not shown). Nevertheless,
expression of several mutant rpoD alleles resulted in
altered levels of in vivo transcription at the
ada promoter: E574A, E575V, I590A, E591A, E605A, and D612A substitutions significantly decreased
ada-dependent transcription, with E575V having
the most severe effect (Fig. 7). Some mutations, such as R596A, K597A,
R608A, and D613A, resulted instead in an increased level of
ada-dependent transcription (120-150%, Fig. 7).

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Fig. 7.
Effects of substitutions in
70CTD on ada-dependent
transcription in vivo. Values are an average of three
independent experiments and are given as a percentage of the wild type.
The average value for the wild type was 903 Miller units.
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To verify that inhibition of ada-dependent
transcription in vivo is indeed due to disruption of
meAda-
70 interaction, we purified both wild
type and E575V
factors and reconstituted RNA polymerase in
vitro. As shown in Fig. 8, E575V
70-RNA polymerase was able to carry out transcription
from the lacUV5 promoter with the same efficiency as wild
type
70-RNA polymerase, which suggests that the E575V
mutation does not affect either core enzyme-
70
interaction or factor-independent transcription; however,
meAda-dependent transcription by the mutant RNA
polymerase at both ada (Fig. 8) and aidB (data
not shown) was drastically impaired.

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Fig. 8.
In vitro transcription with RNA polymerases
reconstituted with either wild type or E575V at the ada
promoter. Fold-activation by meAda is shown below the
transcripts.
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DISCUSSION |
In a previous report, we showed that RNA polymerase binds to the
ada and aidB promoters via the
subunit,
independently of the Ada protein. Thus, meAda does not
recruit RNA polymerase to the promoters but rather converts the RNA
polymerase-promoter complex into a ternary complex proficient in
transcription initiation (17). The location of the meAda
binding site suggests the possibility of interactions with either
CTD or
; alternatively, the mechanism for activation by
meAda could involve turning the
-binding sites of
ada and aidB into more efficient UP elements. Our
experiments with hybrid promoters show that when the UP-like element of
aidB is substituted by the rrnB P1 UP element, no
significant stimulation of transcription occurs (Fig. 2). In contrast,
changing either the
35 or
10 sequences of aidB to
consensus results in a significant stimulation of
ada-independent transcription in vivo, suggesting
that meAda improves RNA polymerase interaction with the
core promoter region (Fig. 2). Thus, meAda activates
transcription either by improving initial binding of RNA polymerase to
the
35 or by facilitating a later step in transcription initiation,
such as isomerization to open complex.
Deletion of
CTD severely affects meAda-activated
transcription (Ref. 16; Fig. 3) and prevents RNA polymerase binding to
the ada and aidB promoters (Fig. 4). However, at
an aidB derivative in which the
10 sequence was changed to
consensus (aidB "
10 con"), meAda is able
to activate transcription by
-235 RNA polymerase (Fig. 3) to roughly
the same extent as wild type RNA polymerase. Thus, dependence on
CTD
for transcription at Ada-dependent promoters can be
by-passed by strengthening the core promoter, presumably by providing
an alternative binding site for
-235 RNA polymerase that compensates
for its loss of interaction with the UP-like element. Although it is
possible that altering the
10 sequence of the aidB
promoter also modifies the interaction between meAda and
RNA polymerase, the results at the aidB
10 con promoter clearly demonstrate that meAda can activate transcription
by RNA polymerase containing an
subunit deleted of its CTD.
Although we cannot rule out the possibility of direct
-meAda interaction, we propose that inefficient
transcription by RNA polymerase deleted of its
CTD results from the
loss of the
-UP-like element interaction and consequent inability of
RNA polymerase to bind the promoter, rather than from lack of
interaction with meAda.
Several lines of evidence show that meAda interacts with
70. Gel retardation experiments (Figs. 5 and 6) indicate
that the terminal 39 amino acids of
70 are necessary for
this interaction. This region contains determinants for the recognition
of the
35 element (region 4.2), followed by the so-called "basic
cluster" (28). Several amino acids in both region 4.2 and in the
basic cluster have been found to be important for interaction with
transcription activators, such as PhoB, bacteriophage
cI protein,
and FNR (29, 30).2 A set of
substitutions between amino acids 570 and 580 of
70
severely impairs transcription activation by PhoB at the
pstS promoter (31) and also affects activation by cI at
PRM promoter (30). Mutational analysis of
70CTD suggests that meAda interacts with a
set of negatively charged residues both in and downstream of region 4.2 (Fig. 7). With the exception of I590A, all the substitutions that
significantly affect ada-dependent transcription
involve negatively charged amino acids (Glu-574, Glu-575, Glu-591,
Glu-605 and Asp-612); substitution to alanine of several positively
charged residues (Arg-596, Lys-597, Arg-608) results in increased
levels of ada-dependent transcription (Fig. 7),
supporting the hypothesis that meAda interacts with a
negatively charged patch in
70 CTD. One of the residues
important for meAda-
70 interaction, glutamic
acid at position 575 (Glu-575), also plays a role in transcription
activation by PhoB (29), suggesting that the location of this residue
allows interaction with different transcription activators. The Glu-575
residue does not seem to be necessary for activator-independent
transcription; neither the E575V mutation described in this report nor
the E575K mutation described in Ref. 29 has any effect on transcription
from the lacUV5 promoter in vitro (Fig. 8), nor
do they affect growth rates in any of the reporter strains tested
(data not shown; Ref. 29).
The specificity of meAda-
70 interaction, the
location of its determinants in the terminal 39 amino acids of
70, and the strong effect of the E575V mutation in
vitro point to
70CTD as the principal target of
activation by meAda at the ada and
aidB promoters. Interestingly, meAda is also
able to activate transcription by
s-RNA polymerase at
both promoters (21, 32).
s is an alternative
factor,
highly expressed in the stationary phase of bacterial growth (33).
s is homologous to
70 in region 4.2 (28),
and the amino acids important for
meAda-dependent transcription are either
conserved residues (Glu-574/Glu-289 and Ile-590/Ile-305) or
conservative substitutions (Glu-575/Asp-290, Glu-591/Gln-306, and
Glu-605/Gln-320) in
s. Thus, it is possible that
meAda could contact amino acids conserved in both
factors; future experiments of site-directed mutagenesis in
s will allow better understanding of activation of
s-dependent transcription by
meAda.
A model for meAda-activation of ada and
aidB is presented in Fig. 9.
In the absence of meAda, RNA polymerase can bind to the
ada and aidB promoters via its
CTD but fails
to establish any strong interaction with the core promoter (17).
meAda does not recruit RNA polymerase to the ada
and aidB promoters, but upon binding of
to the UP
element it interacts with
70 CTD, triggering
transcription initiation; therefore,
subunit-promoter and
meAda-
70 interactions act at separate but
interdependent steps of transcription initiation. It is possible that
Ada CTD is responsible for this interaction; deletions in Ada CTD
abolish transcription activation at the ada promoter without
affecting DNA binding by meAda (14).

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Fig. 9.
Model for transcription activation by
meAda. The upper panel illustrates the
specific interactions established between RNA polymerase and the
ada or the aidB promoters in the absence of
meAda; the RNA polymerase-promoter complex results from
protein-DNA interactions between CTD and the UP elements. The
lower panel shows the RNA
polymerase·promoter·meAda ternary complex.
meAda binds to its DNA site via its N-terminal domain (NTD)
(12) and stimulates transcription initiation (black arrow)
via protein-protein interaction between its C-terminal domain (14) and
70CTD.
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We thank Carol Gross for the gift of the
alanine scan plasmid set, Alicia Dombroski for useful discussion, and
Virgil Rhodius for providing us with the rpoD mutant
library.