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Journal of Bacteriology, October 2000, p. 5539-5550, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Role of the RNA Polymerase
Subunits in
MetR-Dependent Activation of metE and metH:
Important Residues in the C-Terminal Domain and Orientation
Requirements within RNA Polymerase
Paula S.
Fritsch,1
Mark L.
Urbanowski,2 and
George V.
Stauffer2,*
Molecular Biology Graduate
Program,1 and Department of
Microbiology,2 The University of Iowa, Iowa
City, Iowa 52242
Received 23 March 2000/Accepted 7 July 2000
 |
ABSTRACT |
Many transcription factors activate by directly interacting with
RNA polymerase (RNAP). The C terminus of the RNAP
subunit (
CTD)
is a common target of activators. We used both random mutagenesis and
alanine scanning to identify
CTD residues that are crucial for
MetR-dependent activation of metE and metH. We
found that these residues localize to two distinct faces of the
CTD.
The first is a complex surface consisting of residues important for
-DNA interactions, activation of both genes (residues 263, 293, and
320), and activation of either metE only (residues 260, 276, 302, 306, 309, and 322) or metH only (residues 258, 264, 290, 294, and 295). The second is a distinct cluster of residues
important for metE activation only (residues 285, 289, 313, and 314). We propose that a difference in the location of the MetR
binding site for activation at these two promoters accounts for the
differences in the residues of
required for MetR-dependent
activation. We have designed an in vitro reconstitution-purification
protocol that allows us to specifically orient wild-type or mutant
subunits to either the
-associated or the
'-associated position
within RNAP (comprising
2,
,
', and
subunits).
In vitro transcriptions using oriented
RNAP indicate that a single
CTD on either the
- or the
'-associated
subunit is
sufficient for MetR activation of metE, while MetR
interacts preferentially with the
CTD on the
-associated
subunit at metH. We propose that the different
CTD
requirements at these two promoters are due to a combination of the
difference in the location of the activation site and limits on the
rotational flexibility of the
CTD.
 |
INTRODUCTION |
In Escherichia coli and
Salmonella enterica serovar Typhimurium, the final step in
methionine biosynthesis is the transfer of a methyl group to
homocysteine to form methionine. The reaction can be catalyzed by
either of two transmethylases encoded by metE or
metH (12). The metH gene product is
dependent on vitamin B12 as a cofactor, whereas the
metE gene product is not.
Transcription of a number of genes in the methionine biosynthetic
pathway, including metE and metH, is dependent on
the MetR activator protein; MetR is a dimer in solution (25)
and binds to the consensus site 5'-TGAANNTNNTTCA-3'
(49). Using homocysteine as a coactivator, MetR
stimulates expression of the metE promoter up to 200-fold
(48). MetR protects two adjacent sites, from bp
26 to
49
(site 2) and from bp
50 to
73 (site 1) upstream of the
Salmonella serovar Typhimurium metE transcription
start site, from DNase I cleavage (Fig.
1). Within each DNase I-protected region
are perfect (site 1) or near-perfect (site 2) matches to the MetR
consensus binding site sequence centered at
63 and
42 for sites 1 and 2, respectively (49, 54). Although both sites are
necessary for activation of metE, genetic data suggest that site 2 is the activation site; MetR at the high-affinity site 1 appears
to promote the homocysteine-dependent filling by a second MetR dimer at
the lower-affinity site 2 (54). The Salmonella serovar Typhimurium metH gene is activated up to 19-fold by
the presence of MetR (5); however, homocysteine, in contrast
to its role at the metE promoter, decreases this activation
3-fold, probably by an indirect effect of lowering MetR levels
(46, 48). Genetic and biochemical analyses indicate that
there is a single MetR dimer binding site at metH as well as
two alternative start sites separated by 3 intervening bp (Fig. 1)
(5, 47); however, regardless of which start site is used,
the center of the activation site at metH (either at
57 or
at
61) is clearly different from the center of the activation site of
metE at
42. Because of the difference in the number of
MetR binding sites and the location of the activation site at these
promoters, it is possible that MetR may use different mechanisms to
activate these two promoters.

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FIG. 1.
The metE-metR and metH promoters
of Salmonella serovar Typhimurium. Promoter 10 and 35
sequences are boldfaced; those for metE and metH
are overlined, and those for metR are underlined. MetR
binding sequences are lined between the DNA strands. Transcription
start sites are marked with arrows. The metE and
metH transcription start sites were determined by primer
extension (unpublished data). (A) metE-metR promoter region;
(B) metH promoter region.
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Activation at many promoters results from interactions between an
activator protein and RNA polymerase (RNAP) (reviewed in reference
14). The specific subunit of RNAP (
2,
,
', or
) that is contacted by an activator appears to depend
at least in part on the location of the activator binding site relative
to the RNAP binding site. For many activator proteins that bind
upstream of the promoter, such as cyclic AMP receptor protein (CRP),
which binds at the lacP1 promoter, the major contact site on
RNAP is the C terminus of the
dimer subunits (
CTD). The
-CRP
interaction increases the affinity of RNAP for the lac
promoter, which, in turn, increases transcription (reviewed in
reference 8). Recruitment may be a common mechanism
for activators that bind upstream of promoters; it has been shown that
activation can occur when the
CTD is replaced with a heterologous
protein domain capable of interacting with a specific partner protein
bound upstream of the promoter (7). However, the
CTD is
not the exclusive target of activator proteins, and activation may also
be mediated by RNAP-activator interactions that influence steps of
transcription initiation subsequent to RNAP binding. At promoters where
the activator binds to sites adjacent to or overlapping the promoter elements, contacts with the N terminus of
(e.g., CRP at
gal) and with
70 (e.g.,
cI at
PRM) have been reported (23, 31). The phage N4
single-stranded DNA binding protein activates N4 late promoters through
an interaction with the
' subunit, and the
54-dependent activator C4-dicarboxylic acid
transport protein D of Rhizobium meliloti interacts in
solution with both the
subunit and
54 (22,
26).
In this report, we describe experiments aimed at identifying and
understanding the role of activator-RNAP interactions in MetR-dependent
activation of metE and metH. It has previously been shown that removal of the entire
CTD eliminates activation by
MetR at both metE and metH in vitro
(15a). Here we describe the effects of various point
mutations in the
CTD on the activation by MetR at these two
promoters in vivo and in vitro. In addition, we describe a protocol for
the reconstitution of RNAP containing oriented
subunits. We have
used the oriented
RNAPs to show that MetR-dependent activation at
metE and metH have different requirements for the
location of wild-type
subunits within the RNAP enzyme complex in
order for activation by MetR to occur, suggesting that the mechanisms
of activation by MetR differ at these two promoters.
 |
MATERIALS AND METHODS |
Bacterial strains, plasmids, and phages.
The strains and
plasmids used in this study are listed in Table
1.
Plasmid pREII-Strep

was constructed using site-directed mutagenesis
to insert the sequence 5'-GCT TGG AGC CAC CCG CAG TTC
GAA AAA GGT
GCT-3' (encoding the
Strep-tag II amino acid sequence
WSHPQFEK [
37] flanked by an alanine on the
5' end and a glycine
and an alanine on the 3' end) between codons 1 and
2 of the
rpoA gene in plasmid pREII

. For production of
Strep-tagged

protein,
plasmid pT7-Strep

was constructed by
replacement of the
XbaI-
BamHI
fragment of
pHTT7f1-NH

with the corresponding fragment from pREII-Strep

.
This
plasmid did not overproduce the Strep-tagged

as well as
plasmid
pHTT7f1-NH

overproduces the His
6-tagged

(data not
shown);
however, we could purify enough Strep-tagged

for
reconstitutions
of oriented

RNAP. Plasmid pT7-Strep(R45A)

was
constructed by
site-directed mutagenesis to convert codon 45 of
rpoA from a CGT
(arginine) to a GCT (alanine) codon in
plasmid pT7-Strep

. Plasmid
pHTT7f1-NH(

CTD)

was constructed by
site-directed mutagenesis
to convert codon 257 of the
rpoA
gene in plasmid pHTT7f1-NH

from
GTT (valine) to TAA (stop) and
create a
BamHI restriction site
following the new stop
codon. Plasmid pT7-Strep(R45A/

CTD)

was
constructed by replacement
of the
HindIII-
BamHI fragment of
pT7-Strep(R45A)
with the corresponding fragment from
pHTT7f1-NH(

CTD)

. The plasmids
used to express the
His
6-tagged N268D, L270H, and G296A

subunits
were
generated by replacing the
HindIII-
BamHI
fragments of pHTT7f1-NH
with the corresponding fragments from
pREII268D

, pREII270H

,
and pREII296A

. The
His
6-tagged R265A derivative was generated
by replacing the
HindIII-
BamHI fragment of pHTT7f1-NH

with
the
HindIII-
BstYI fragment of pHTf1265A

.
The constructs were confirmed
by DNA
sequencing.
The
TElac1 phage, a

gt2 derivative containing a
metE-lacZ translational fusion, is a temperature-resistant
derivative of

Elac1, which has been previously described
(
33). The

Hlac
phage, also a

gt2 derivative but
carrying a
metH-lacZ translational
fusion, has been
described previously (
47).
Media and growth conditions.
Tryptone broth (TB),
Luria-Bertani broth (LB), and lactose tetrazolium agar were prepared as
described previously (27). Glucose minimal medium was Vogel
and Bonner minimal salts (51) supplemented with 0.4%
glucose. Minimal medium was also supplemented with phenylalanine (50 µg/ml) and thiamine (1 µg/ml), since most of the strains carry the
pheA905 and thi markers. Strains carrying
plasmids were maintained in medium supplemented with ampicillin at 100 µg/ml; however, transformants used for the production of RNAP
subunits were supplemented with ampicillin at 200 µg/ml. Strains
carrying pGS395 were maintained in medium supplemented with kanamycin
at 20 µg/ml.
Lysogens were grown at 37°C, except for

Hlac lysogens, which were
grown at 30°C, since the

Hlac phage carries the
cI857,
mutation resulting in a temperature-sensitive

cI repressor (
32).
-Galactosidase enzyme assays.
For all strains except
those carrying the rpoA112 allele, cells were grown in TB to
an optical density at 600 nm (OD600) of approximately 0.5 and then placed on ice for 20 min. The rpoA112 strain GS1106
was grown as 2- by 2-cm patches on LB agar overnight at the desired
temperature; prior to being assayed, the cells were scraped from the
plate, resuspended in 1× Vogel and Bonner minimal salts to an
OD600 of approximately 0.5, and then placed on ice for 20 min.
-Galactosidase enzyme activity was assayed using the
chloroform-sodium dodecyl sulfate (SDS) lysis procedure (27). Assays were performed at least twice, with the
activity of each sample determined in triplicate.
Mutant isolation. (i) Selection for mutations that affect
metE expression.
Random mutagenesis of the 1-kb
XbaI-BamHI rpoA fragment from pREII
was performed as described previously (42). The
PCR-mutagenized rpoA gene was cloned into pREII
in place
of the wild-type XbaI-BamHI fragment. The
selection strain, GS972
TElac1, forms white colonies on
lactose tetrazolium agar when transformed with pREII
. Transformants
of GS972
TElac1 with decreased expression of the
metE-lacZ fusion were identified as red colonies on lactose
tetrazolium agar. The mutations were further mapped to the
CTD by
subcloning the HindIII-BamHI fragment (codons
230 to 329) from the mutant plasmids into pREII
.
(ii) Screen for mutations that affect metE and/or
metH expression.
Plasmids from an alanine substitution
library of the
CTD (residues 255 to 329) were individually
transformed into GS162
TElac1 and GS972
Hlac. As a
preliminary screen, a single colony of each transformant was grown for
3 h (OD600 of approximately 0.5) in TB and assayed for
-galactosidase activity. Transformants that showed decreases in
either metE-lacZ or metH-lacZ expression were
selected for further study. Eight of the alanine substitutions increased metE expression in the preliminary screen (maximum
metE-lacZ expression was 2.3-fold higher than that in
wild-type
). However, these mutants were not included in this study.
Preparation of core RNAP containing
homodimers.
The
His6-tagged
subunits were prepared under denaturing
conditions as previously described (40) with the following
modifications: cells were lysed by sonication in buffer B (6 M
guanidine-HCl, 20 mM Tris-HCl [pH 7.9], 500 mM NaCl, 5 mM imidazole)
instead of a nondenaturing buffer, and the ammonium sulfate
precipitation step was eliminated. Inclusion bodies containing
and
' were prepared as described previously (40).
Reconstitutions were performed as described previously (40)
with the following exceptions: reconstitution of
70 was
not performed at the same time as the core RNAP reconstitution, and
activation of the RNAP following dialysis was performed in the absence
of
70. Following activation, the core RNAP mixture was
cleared by centrifugation and the reconstituted RNAP was purified by
Ni2+ ion affinity chromatography as described previously
(40). The resulting sample was concentrated to approximately
50 µl with a Microcon YM-100 concentrator (Millipore, Bedford,
Mass.), mixed with an equal volume of glycerol, and stored at
20°C.
Preparation of Strep-tagged
.
E. coli strain
BL21(DE3) transformed with pT7-Strep
(or derivatives) was shaken at
37°C in 100 ml of LB plus ampicillin (200 µg/ml) to an
OD600 of approximately 0.5, induced by the addition of
isopropyl-
-D-thiogalactoside (IPTG) to 1 mM, and shaken
an additional 6 to 15 h at 22°C. The cells were harvested by
centrifugation (4,500 × g; 12 min at 4°C) and
resuspended in 1.5 ml of buffer W (100 mM Tris-HCl [pH 8.0]-1 mM
EDTA) at 4°C. Cells were lysed by sonication, and the lysate was
cleared by centrifugation (16,000 × g; 15 min at
4°C). The supernatant was treated with 7 µl of a 1-mg/ml avidin
solution (prepared in buffer W) for 30 min at 4°C and cleared by
centrifugation (16,000 × g; 15 min at 4°C). The
extract was adsorbed to a 1-ml StrepTactin column (Genosys Biotechnologies, Woodlands, Tex.) preequilibrated with buffer W, washed
five times with 1 ml of buffer W, and eluted six times with 0.5 ml of
buffer E (buffer W with 2.5 mM desthiobiotin). The bulk of the protein
eluted in one fraction; the yield was 100 µg of protein as determined
by the Bradford assay (4), and the purity was >50% as
determined by SDS-polyacrylamide gel electrophoresis (PAGE). The
contaminating proteins appeared, based on size, to be the other RNAP
subunits; however, native, non-tagged
is distinguishable from the
Strep-tagged
on our SDS-15% PAGE gels, and we could not detect
any contaminating non-tagged
by staining with Coomassie brilliant
blue R.
Preparation of core RNAP containing oriented
subunits.
,
', and His6-tagged and Strep-tagged
subunits
were prepared as described above. For each reconstitution, 30 µg of
each
species was added to make 60 µg of total
. Reconstitution
and a first partial purification of core RNAP by Ni2+ ion
affinity chromatography based on the His6 tag were
performed as described above for the purification of core RNAP
containing
homodimers. The eluate from the
Ni2+-nitrilotriacetic acid resin (Qiagen Inc., Valencia,
Calif.) following this first purification was then applied to a
StrepTactin column (bed volume, 1 ml). Adsorption, washes, and elution
of the StrepTactin column were performed as described above for the
purification of Strep-tagged
. The RNAP typically eluted in one or
two fractions that were concentrated as described above for the
purification of core RNAP containing
homodimers. The yield was
typically 5 to 10 µg, which is much lower than what is recovered from
the single purification step for homodimeric RNAP (40);
however, these yields were sufficient for several transcription
experiments. The concentrated sample contained no traces of native,
non-tagged
, as determined by Coomassie brilliant blue R staining of
an SDS-15% PAGE gel (data not shown).
In vitro transcription assays.
Aliquots of reconstituted
core RNAP were incubated in the presence of a 1.5 molar excess of
purified
70 subunit to ensure the formation of RNAP
holoenzyme for use in single-round runoff transcription assays in the
presence of heparin as previously described (16). The
activity of each reconstituted RNAP was normalized based on the amounts
of lacUV5 and RNA-I transcripts produced when the
supercoiled plasmid pRLG593 was used as the template (35).
Based on these initial experiments, the enzymes were used at the
concentrations indicated in Fig. 3A and Fig. 4A to test the effect of
RNAP containing mutant homodimers or oriented heterodimers of
on
metE and metH transcription in the absence or
presence of MetR (final dimer concentration, 135 and 270 nM). Template
DNA containing both the metE and metR promoters and the metH template DNA were added simultaneously to
reaction mixtures when the RNAPs containing homodimers of
were
tested. In transcriptions with the oriented
RNAP, the
metE-metR and metH templates were tested
separately. Following electrophoresis, the gels were dried and analyzed
by autoradiography. When quantitated, the bands corresponding to the
metE and metH transcripts were analyzed with a
Packard InstantImager (Meriden, Conn.). For reporting, the quantitated
amount of metE and metH transcript produced by each RNAP was normalized to the sum of the lacUV5 and RNA-I
transcripts generated from the supercoiled template pRLG593
(35) by the same RNAP. Results for
homodimer RNAP are
averages from two transcription experiments. Oriented
RNAP results
are averages from two transcription experiments for metE and
three transcriptions for metH.
 |
RESULTS |
Selection for
mutations that decrease metE
expression.
A plasmid-borne rpoA gene was randomly
mutagenized for use in a genetic screen that would identify colonies
with decreased metE expression. The strain used for the
selection, GS972, constitutively expresses MetR. The overexpression of
MetR was necessary because the rich medium used for the selection
(lactose tetrazolium agar) would repress the wild-type metR
promoter. A
lysogen of GS972 carrying a metE-lacZ
fusion, GS972
TElac1, when transformed with plasmid
pREII
expressing wild-type rpoA, produces white colonies
on lactose tetrazolium agar. Although the chromosomal rpoA
gene is present in this and all of the transformants used in this
study, it has been shown by Western blotting that the
proteins
expressed from the high-copy-number plasmids are the predominant
species in the cell (42). Screening of three independent
plasmid pools carrying PCR-mutagenized rpoA genes yielded
two transformants that formed red colonies on lactose tetrazolium agar,
indicating decreased metE-lacZ expression. The metE-down phenotype of one of the mutants was shown to be
plasmid associated and could be localized to the
CTD by subcloning
of the rpoA gene. Sequencing of this mutant revealed a
single-base-pair substitution that resulted in a change from asparagine
to aspartic acid at amino acid 268 (N268D
). For the second mutant, a
plasmid association test revealed that the metE-down
phenotype was unstable in GS972
TElac1, and although
subcloning showed that the phenotype was associated with the
CTD, the subclones were also unstable in GS972
TElac1.
Parallel subcloning of this mutant in GS162
TElac1, the
wild-type parent of GS972 carrying the metE-lacZ fusion, eventually gave rise to a stable phenotype by
-galactosidase assays
(data not shown). Sequencing of this stable mutant revealed a
single-base-pair substitution that resulted in a change from leucine to
histidine at amino acid 270 (L270H
).
To quantitate the effects of these

CTD mutations on
metE
expression, GS162
TElac1 was transformed with plasmids
expressing either wild-type

or one of the mutant

alleles. These
transformants were grown
in TB, and

-galactosidase levels were
determined. As expected,
the N268D and L270H substitutions strongly
decreased
metE-lacZ expression (Table
2). The
metE-down phenotype
associated with
these mutations was lost if the mutant alleles were
expressed
in GS244
TElac1, a
metR strain,
suggesting that these

CTD mutations disrupt
MetR-dependent
expression of
metE instead of causing a general
defect in
transcription (Table
2).
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TABLE 2.
Effects of point mutations at amino acids 268 and 270 of
the subunit on metE-lacZ and
metH-lacZ expression
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|
Following multiple manipulations, including plasmid preparation and
subcloning, the N268D and L270H
rpoA alleles were assayed
in
the retransformed lysogen GS972
TElac1. The N268D
substitution decreased
metE expression only threefold,
compared to a ninefold decrease in GS162
TElac1.
Surprisingly, however, the L270H phenotype was completely
suppressed
(Table
2), so it is unclear how the L270H mutant was
isolated as a red
colony on lactose tetrazolium plates. We speculate
that the original
isolate, which exhibited a stronger phenotype
on lactose tetrazolium
plates than the N268D mutant, may have
carried a second mutation (or an
alternate substitution at amino
acid 270) that was lost because of the
detrimental effect it had
on overall cellular gene expression. We
further speculate that
the second mutation was in the

CTD because
the phenotype transferred,
although unstably, with the

CTD
subclones.
The N268D and L270H mutations were also tested for their effects on
metH-lacZ expression. The same three strains used to test
the effects on
metE-lacZ expression were lysogenized with a
phage
carrying a
metH-lacZ fusion. In GS162

Hlac, neither

mutation
caused a decrease in
metH-lacZ expression.
However, in GS972

Hlac,
the N268D substitution caused nearly a
threefold decrease in
metH-lacZ expression while the L270H
substitution yielded essentially wild-type
levels (Table
2). The effect
of the N268D substitution on
metH expression is MetR
dependent, because the phenotype was lost in
a
metR
background (Table
2).
Screen for alanine substitutions in the
CTD that alter
metE and/or metH expression.
To more
efficiently identify residues in the
CTD important for activation of
metE and/or metH, we switched to screening an alanine substitution plasmid library of the
CTD (residues 255 to 329). Lysogens GS162
TElac1 and GS972
Hlac
were transformed with the plasmid library to assess the effect of each
alanine substitution on both metE-lacZ and
metH-lacZ expression. Transformants that showed decreases in
either metE-lacZ or metH-lacZ expression in a
preliminary screen were selected for further study.
Many of the alanine substitutions had modest effects on
metE
expression (Fig.
2A). Among the residues
that, when changed to
alanine, cause at least a twofold decrease in
metE expression
are the previously characterized DNA binding
residues of

: L262,
R265, N268, C269, G296, K298, and S299 (
10,
17,
28). Consistent
with the selection using PCR mutagenesis,
L270 was also identified
as important for
metE expression in
this screen. In addition,
alanine substitutions at residues L260, T263,
H276, I278, L281,
L289, P293, E302, I303, V306, L307, S309, S313, L314,
N320, and
P322 all cause at least twofold decreases in
metE-lacZ expression,
with changes at E302, I303, and L314
being more severe than changes
at most of the DNA binding residues. The
alanine substitution
mutants that showed a
metE-down
phenotype in GS162
TElac1 were also tested in
GS244
TElac1 to determine the effects of the alanine
substitutions on
basal
metE expression. With the exception
of I303A, the alanine
substitutions caused at most a 1.3-fold down
effect on
metE-lacZ expression in the absence of MetR (data
not shown). These results
suggest that the
metE-down
phenotypes caused by these alanine
substitutions are MetR dependent.
The I303A substitution caused
a twofold reduction in basal
metE-lacZ levels (data not shown).
Since this substitution
caused a fivefold reduction in MetR-dependent
metE
expression (Fig.
2A), the I303A substitution appears to have
an effect
on both basal and activated transcription of
metE. Based
on
the solution structure of the

CTD (
10,
17), all but five
of these residues (L270, I278, L281, I303, and L307) are surface
exposed; therefore, these residues could contact MetR for activation.

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FIG. 2.
Alanine scan of CTD. Plasmids expressing
rpoA with single alanine substitutions at each residue in
the CTD (residues 255 to 329) were assayed for effects on chromosomal
metE-lacZ (A) and metH-lacZ (B) expression.
Results are expressed as percentages of the activity in cells carrying
plasmids expressing the wild-type rpoA gene. Residues 267, 272, 274, 308, 324, and 327 (filled bars) are alanines in the wild-type
protein. Residues with no data did not decrease expression of either
gene in an initial screen.
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|
Far fewer

CTD alanine substitutions cause at least twofold decreases
in
metH expression (Fig.
2B). Those that do, R265, N268,
K298, and S299, are all DNA binding residues of

. However, based
on
in vitro results, using a twofold decrease in expression as
a cutoff
for determining whether a residue is important for
metH expression may be too stringent (see below). Alanine substitutions
at
D258, L262, T263, V264, C269, L270, L290, P293, N294, L295,
G296, I303,
and N320 reproducibly cause 1.3- to 1.9-fold decreases
in
metH expression. These 17 alanine substitution mutants were
also tested in GS244

Hlac to determine the effect of the loss
of MetR
on the phenotype (data not shown). The
metH-down phenotype
of all of these mutants was lost in the
metR background,
suggesting
that these alanine substitutions cause decreases in
MetR-dependent
expression of
metH. All but two of these
residues (L270 and I303)
are surface exposed (
10,
17);
therefore, these residues could
contact MetR for
activation.
In vitro analysis of
CTD mutants.
Many of the
CTD
mutations show some MetR-dependent phenotype in vivo, especially at
metE. However, some of these phenotypes may be due to
indirect effects, e.g., altering levels of the activator or levels of
the enzymes involved in homocysteine metabolism. To determine the
direct effect of
CTD mutations on MetR-dependent expression, we
reconstituted RNAP in vitro in the presence of either
His6-tagged wild-type or mutant
subunits for use in an in vitro transcription system.
Four

mutants were tested in vitro: three substitutions in

DNA
binding residues (R265A, N268D, and G296A) and L270H, since
this
substitution affects
metE but not
metH expression
in vivo.
The activity of each reconstituted RNAP was normalized based
on
its ability to make the

CTD-independent
lacUV5 and
RNA-I transcripts
from plasmid pRLG593 (Fig.
3A) (
34).

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FIG. 3.
In vitro transcriptions using RNAP with CTD
substitutions. The RNAP is designated by the subunits in the
holoenzyme. (A) Transcripts from single-round transcription experiments
with the supercoiled plasmid pRLG593 as a template to equalize the
activity of each RNAP. Indicated are the positions of the
CTD-independent transcript initiated at the lacUV5
promoter and the plasmid-derived RNA-I transcript. The activity of the
G296A RNAP was determined in a separate experiment, so it is shown with
the wild-type RNAP from the same experiment. RNAPs were used at the
following concentrations in these experiments: 20 nM wild-type, R265A,
and N268D RNAPs, 60 nM L270H RNAP, and 50 nM G296A RNAP. (B)
Transcripts from single-round transcription experiments using a linear
template carrying the metE-metR control region as well as a
linear template with the metH promoter in the absence ( )
and the presence of 135 nM (+) or 270 nM (++) MetR dimer. The
transcripts initiated at the metE and metH
promoters are indicated. (C and D) Effects of CTD substitutions on
metE (C) and metH (D) activation. The
metE transcript produced by each RNAP in the presence of 135 nM MetR and the metH transcript produced by each RNAP in the
presence of 270 nM MetR from two independent in vitro transcription
experiments were quantitated and normalized to the amounts of
lacUV5 and RNA-I transcripts produced by the same RNAP. The
normalized amount of transcript produced by each mutant RNAP is
reported as a percentage of the expression by wild-type RNAP (± 1 standard deviation).
|
|
In a purified transcription system containing template DNA,
homocysteine, ribonucleoside triphosphates, and wild-type RNAP,
both
metE and
metH expression were dependent on the
addition of
MetR (Fig.
3B, lanes 7 to 9). However, when the mutant
RNAPs were
used, all four

mutations caused severe decreases in
MetR-dependent
metE expression (Fig.
3B [compare lane 8 to
lanes 11, 14, and
17, and compare lane 20 to lane 23] and 3C).
Quantification of
metE levels in the absence of MetR
indicates that all four of
these mutations in the

CTD caused less
than 2.5-fold decreases
in basal
metE expression (Fig.
3B,
lanes 7, 10, 13, 16, 19, and
22); however, this quantification is
subject to large errors because
the
metE basal levels are so
low. As indicated above, the in vivo
metE-down phenotype of
most of the

CTD substitutions, including
R265A, N268D, L270H, and
G296A, was lost in the absence of MetR,
suggesting that these mutations
primarily affect activated
metE transcription.
The effects of the

CTD mutations on
metH were also tested
in the same transcription reactions. All four

mutations resulted
in
severe defects in MetR-dependent activation of
metH
expression
(Fig.
3B [compare lane 9 to lanes 12, 15, and 18, and
compare
lane 21 to lane 24] and 3D). The L270H substitution had only a
minor effect on
metH expression in vivo (Table
2); however,
in
the in vitro system, the effect of this mutation on
metH
expression
was as severe as those of the other point mutations (Fig.
3D).
As is the case for
metE, quantification of the basal
metH levels
is subject to large errors; however, if we
measure these levels,
none of the substitutions caused more than a
1.6-fold decrease
in basal
metH expression. These in vitro
results are consistent
with our in vivo analysis of basal
metH levels, where we found
that none of the

CTD
substitutions that affected
metH levels
in the presence of
MetR had any effect on
metH levels in the absence
of
MetR.
In vivo, the
metE-down phenotypes of all four of these
mutations could be at least partially suppressed by the high
constitutive
MetR levels in GS972 (Table
2 and data not shown). This is
not
the case in vitro, however. As more MetR was added to reaction
mixtures (270 nM dimer) with wild-type RNAP, the amount of
metE transcript decreased (Fig.
3B; compare lanes 8 and 9).
The mutant
RNAPs also produced less
metE transcript in the
presence of 270
nM MetR dimer than with 135 nM MetR (Fig.
3B; compare
lanes 11
and 12, 14 and 15, 17 and 18, and 23 and 24). These results
suggest
that the suppression seen in vivo is due to the presence of
chromosomally
derived wild-type

that is incorporated into
RNAP.
A wild-type
CTD in the
'-associated
subunit is sufficient
for activation of metE in vivo.
The in vivo and in
vitro experiments show significant differences in the magnitudes of the
effects of the mutant
CTDs on activation, the effect of the L270H
substitution on metH expression, and the suppression of the
metE phenotypes by high concentrations of MetR. We
considered the possibility that these differences were due to the
nature of the mutant RNAPs used in vivo and in vitro. In vivo, even
though the mutant rpoA genes expressed from high-copy-number
plasmids should be the predominant form of
in RNAP, the chromosomal
rpoA is intact, so wild-type
can also be incorporated
into RNAP either as homodimers of wild-type
or heterodimers of
wild-type and mutant
. In contrast, the in vitro purification and
reconstitution protocol produces wild-type or mutant RNAP having
dimers of homogeneous subunits. In an attempt to reproduce in vivo the
severe loss of MetR activation observed in vitro, we constructed a
derivative of the wild-type GS162
TElac1 lysogen,
GS1106
TElac1, in which the chromosomal copy of
rpoA was replaced with the rpoA112 allele
encoding an
subunit reported to have a temperature-sensitive lethal
defect for assembly of core polymerase (20). Strains carrying rpoA112 on the chromosome cannot grow at the
nonpermissive temperature (42°C) unless an alternative functional
rpoA gene is also present, such as a plasmid-borne
rpoA gene. However, certain
CTD substitution mutations
are unable to complement the rpoA112 allele at 42°C,
including R265A, N268A and G296A (10). The L270H
allele
can complement rpoA112, so the effect of L270H
on
metE-lacZ was examined in the rpoA112 background.
Since expression of the metR promoter is severely reduced at
high temperatures (data not shown), the cells used for the assay also
carried plasmid pGS395, which constitutively expresses MetR from a
heterologous promoter.
As expected based on the results in GS972
TElac1, when
tested in the
rpoA112 background at a permissive temperature
(37°C)
that allows the RpoA112

protein to participate normally in
the
assembly of RNAP, the L270H substitution did not affect
metE-lacZ expression (Table
3). Surprisingly, when tested at the
nonpermissive
temperature (42°C) that inhibits normal assembly by the
RpoA112

protein, the cells expressing the L270H

did not mimic
the severe
loss of MetR activation predicted from the in vitro results
if
all of the cellular RNAP at 42°C had incorporated homogeneous
L270H

dimers; instead, these cells expressed
metE-lacZ as
well
as did the wild type (Table
3).
The
rpoA112 assembly defect at the nonpermissive temperature
is due to an arginine-to-cysteine substitution at position 45
of

(R45C

) (
15). An alanine substitution at the same

position
(R45A

) can still dimerize but can no longer interact with
the

subunit even at 37°C to assemble the
2
precursor required
for core polymerase formation (
21).
However, mixtures of R45A
and wild-type

can form

heterodimers in vitro that successfully
interact with the

subunit
via the wild-type moiety and can thus
form core polymerase having
oriented

subunits: wild-type

contacting
the

subunit
(
I) and R45A

contacting the

' subunit
(
II) (
29). If the consequence of the
rpoA112 mutation (R45C

) at
the nonpermissive temperature
is the same as that of the R45A
substitution, then cultures of the
rpoA112 lysogen GS1106
TElac1 expressing the
plasmid-encoded L270H

and grown at 42°C
should produce a fraction
of RNAP that contains L270H

homodimers,
as well as some RNAP that
contains mixed L270H
I-R45C
II
heterodimers. The homodimeric L270H

RNAP should be equivalent
to the
purified His
6-tagged L270H

RNAP used in the in vitro
transcription
experiments and would thus contribute very little to
metE-lacZ expression (Fig.
3C). According to this
hypothesis, the nearly
wild-type level of expression of
metE-lacZ in the
rpoA112 background
cell must be
due principally to the heterodimeric
L270H
I-R45C
II RNAP. Furthermore, these
results would predict that MetR activation
of
metE is
insensitive to the residue at position 270 of
I provided
that the wild-type residue is present on
II.
The equivalent in vivo experiment to address the effect of oriented

RNAP on
metH expression is technically not possible.
The
rpoA112 allele requires a temperature of at least 42°C for
the orienting phenotype; unfortunately, the
metH promoter is
itself
temperature sensitive and shows virtually no expression at
42°C
(data not
shown).
Orientation requirements of a wild-type
CTD within RNAP for
MetR-dependent activation of metE and metH.
Since the results above suggest that a point mutation in the CTD of
I does not interfere with MetR-dependent activation of
metE, we wanted to test whether an RNAP with a mutation in
the CTD of
II (and a wild-type
I) would
also activate metE, i.e., whether a single wild-type
CTD is sufficient for activation at metE. We also wanted to know
what the requirements for the orientation of
were for activation at
the metH promoter, since we were not able to test the effect of the L270H
I-R45C
II RNAP on
metH in vivo due to the temperature sensitivity of the metH promoter. Although
-orienting substitutions
(L48A, K86A, and V173A) in
analogous to the
'-orienting
R45A substitution have been described (21), simple in vivo
assays with such mutants are not possible because conditional,
chromosomal versions of the
-orienting mutants are presently not
available. We therefore designed an in vitro
reconstitution-purification protocol to purify RNAP containing oriented
subunits. This protocol is an extension of the scheme developed by
Tang et al. (40), which involves purification of RNAP from
an in vitro reconstitution mixture by Ni2+ ion affinity
chromatography based on a His6-tagged
. We included in
the reconstitution mixture a second source of
that carries an
alternate affinity tag, allowing sequential purification of RNAP based
on both tags, ensuring that the final RNAP incorporates an
dimer
composed of two differently tagged monomers. Furthermore, by
specifically including the R45A substitution in one of the two
differently tagged
monomers, we can direct this monomer to the
II position, and, by default, direct the other monomer
to the
I position. Thus,
CTD mutations can be
incorporated into either the
I- or
II-specific monomers to test the orientation-dependent
effects of the
CTD mutations in vitro. We constructed a plasmid,
pREII-Strep
, that expresses an
containing the
Strep-tag II sequence (Strep tag) between codons 1 and 2 of
rpoA, the same location as the N-terminal His6
tag in pHTT7f1-NH
(see Materials and Methods). It has been
previously shown that incorporation of a His6 tag into the
N terminus of
does not impair enzyme function (41). To
determine whether the Strep-tagged
is also functional, we tested
for the ability of Strep-tagged
to complement a known rpoA mutant in vivo. The mutant strain GS1040 carries the
chromosomal E261K rpoA allele, which prevents growth on
glucose minimal medium (16). Transformants of GS1040
carrying the pREII-Strep
plasmid exhibit growth on glucose minimal
medium indistinguishable from that of the wild-type strain (GS162) or
from that of transformants of GS1040 carrying pREII
, suggesting that
Strep-tagged
is incorporated into RNAP and that the Strep tag does
not interfere with function.
Since neither an N-terminal His
6 tag nor a Strep tag
interferes with RNAP assembly or function, purified His
6-
and Strep-tagged

were added simultaneously to the reconstitution
mixture. Incorporation
of the R45A substitution into the Strep tag

construct ensures
that this monomer could be incorporated into RNAP
only at the
II position. Following
renaturation-reconstitution, the RNAP was
purified in two steps, first
based on Ni
2+ ion affinity of the His
6-tagged

subunit, then based on StrepTactin
affinity of the Strep-tagged

subunit, generating a single population
of RNAP containing oriented

subunits. The C-terminal domains
of these
rpoA plasmids can
be altered to generate a set of

CTD
mutant RNAPs with oriented

subunits. For this analysis, we chose
to use a version of

in which
the final 73 amino acids were deleted
(
CTD
) instead
of the L270H mutant

, because the effect
of substitutions at L270
may be indirect (see
Discussion).
Using this reconstitution and purification protocol, we generated four
different RNAP species to test the positional requirements
of the

CTD for MetR-dependent activation in vitro. The first
RNAP contained

subunits with both

CTDs intact (wt
I-wt
II); in the second RNAP, the final 73 amino acids
of

were deleted
from both

subunits
(
CTD
I-
CTD
II);
in the third and fourth RNAP species, the

CTD was deleted
from
either the

-associated
I
(
CTD
I-wt
II) or the

'-associated
II (wt
I-
CTD
II). The activity of
each RNAP was normalized to the wild-type enzyme
using

CTD-independent promoters (Fig.
4A),
and the effect of
deleting each

CTD on
metE and
metH expression was examined. As
expected from previous
studies (
15a), MetR-dependent activation
of
metE
transcription seen with the wild-type RNAP was almost
completely lost
when both

CTDs were deleted (Fig.
4B; compare
lanes 6 and 12).
However, both single-

CTD RNAP derivatives,
CTD
I-wt
II and wt
I-
CTD
II, responded to
MetR-mediated activation nearly as well (50% ±
2% and 70% ± 2%,
respectively) as the wild-type RNAP (Fig.
4B,
lanes 6, 8, and 10).
These results indicate that a single wild-type

CTD in either the
I or
II position of RNAP is sufficient
for MetR-dependent activation
of
metE.

View larger version (25K):
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|
FIG. 4.
In vitro transcriptions using RNAP with oriented subunits. The RNAP is designated by the subunits in the holoenzyme.
(A) Transcripts from single-round transcription experiments with the
supercoiled plasmid pRLG593 as a template to equalize the activity of
each RNAP. Indicated are the positions of the CTD-independent
transcripts initiated at the lacUV5 promoter and the
plasmid-derived RNA-I transcript. The RNAPs were used at the following
concentrations in these experiments: 10 nM wt I-wt
II and CTD I-wt
II RNAPs, and 13 nM wt
I- CTD II and
CTD I- CTD II
RNAPs. (B) Transcripts from single-round transcription experiments
using a linear template carrying the metE-metR control
region in the absence ( ) and presence (+) of 135 nM MetR. The
transcript initiated at the metE promoter is indicated. (C)
Transcripts from single-round transcription experiments using a linear
template carrying the metH promoter in the absence ( ) and
presence (++) of 270 nM MetR. The transcript initiated at the
metH promoter is indicated.
|
|
The oriented

RNAPs were also tested for their effects on
MetR-dependent expression of
metH in vitro. Consistent with
previous
results (
15a), the
CTD
I-
CTD
II
RNAP derivative did not show any appreciable increase in
metH transcription in the presence of MetR (Fig.
4C; compare
lanes
13 and 14 to lanes 19 and 20). Consistent with its activity at
the
metE promoter, the RNAP derivative having a deletion of
the

CTD only at
II responded to MetR activation of
metH 47% ± 3% as well as the
wild-type RNAP (Fig.
4C,
lanes 14 and 18). In contrast, the RNAP
derivative having a deletion of
the

CTD only at
I responded poorly to MetR activation
of
metH, showing only 21%
± 3% of the levels seen with
the wild-type RNAP (Fig.
4C, lanes
14 and 16). These results indicate
that MetR exhibits a more stringent
requirement for a functional CTD on
the

-associated
I subunit than for the CTD of
II for activation of
metH.
 |
DISCUSSION |
Amino acids in the
CTD important for MetR activation of
metE.
Through random PCR mutagenesis of rpoA we
have identified two residues, N268 and L270, that are important for
MetR-dependent activation of metE. Residue N268 has
previously been characterized as a DNA binding residue of
(10,
17, 28). The N268D substitution has also been shown to affect
CRP- and OxyR-dependent activation of the lacP1 and
katG promoters, respectively (44, 57). An alanine
substitution at position 268 not only disrupts activation at promoters
containing an UP element (10, 28), where the
CTD contacts
the DNA directly, but also has been found to decrease activation at
nearly every native promoter tested that requires the
CTD for
activation (for examples, see reference 30).
Amino acid L270 is not an

-DNA interaction residue; however, an
L270A substitution has been shown to disrupt CRP-dependent
activation
at
lacP1 and TyrR-mediated activation of
mtr to
nearly
the same extent as substitutions in

-DNA binding residues
(e.g.,
R265A) (
28,
55). In addition, an L270P substitution
in

disrupts
CRP activation of
lacP1 and CysB activation
of
adi (
39,
57).
As previously suggested by
Murakami et al. (
28), the effect
of a mutation at 270 may
not define a point of contact between

and activator proteins but
instead may be indirect. Amino acid
270 is located in helix 1 of the

CTD, which also contains three
of the DNA binding residues (R265,
N268, and C269) (
10,
17);
therefore, substitutions at 270 may disrupt the structure of helix
1, thereby altering a portion of the

-DNA binding
surface.
The N268D and L270H substitutions are interesting in that the
metE phenotypes of these mutations can be partially to
completely
suppressed by high levels of the activator in vivo. The
phenomenon
of activator overexpression suppressing an
rpoA
phenotype is not
novel. In
Salmonella serovar Typhimurium,
if the FNR homologue
OxrA is overexpressed, the phenotype of the
rpoA8 mutation (G311R

)
is partially suppressed
(
24). However, suppression of the N268D
and L270H phenotypes
is not observed in the in vitro transcriptions
where purified RNAP
containing N268D

or L270H

is used (Fig.
3B), suggesting that when
MetR levels are high in vivo, wild-type

RNAP will be preferentially
recruited to
metE.
Screening of an

CTD alanine substitution library identified
additional residues that are important for
metE activation
by
MetR. Most of the surface-exposed residues identified in this
screen
cluster to a complex face of

that includes residues important
for
activation of both
metE and
metH (Fig.
5). Residues L262,
R265, N268, C269,
G296, K298, and S299, which have previously
been identified as DNA
binding residues of

(
10,
17,
28),
localize to this
complex face. It is possible that these residues
define an interaction
surface on

for contact with MetR because
they affect MetR-dependent
activation of
metE in vivo; however,
we favor the alternate
hypothesis previously proposed for these
residues in CRP-, Mor-, and
Ogr-dependent activation (
1,
3,
10,
36,
53): L262, R265,
N268, C269, G296, K298, and S299
are involved in nonspecific
protein-DNA interactions that stabilize
the activator-

interaction.

View larger version (59K):
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|
FIG. 5.
The solution structure of the CTD (17),
showing the positions of residues identified as important for
MetR-dependent activation at metE and metH in
vivo and, in some cases, verified in vitro. Red, residues important for
both metE and metH expression; yellow, residues
found to be important for MetR-dependent expression of metE
only; blue, residues found to be important for MetR-dependent
expression of metH only. Views (i) to (iv) are related by
90° rotations about the vertical axis.
|
|
Residues L260, T263, H276, P293, E302, V306, S309, N320, and P322 also
localize to this complex face of

, with H276, N320,
and P322 forming
an outlying extension of the face and the others
situated near the DNA
binding residues (Fig.
5). Since the phenotype
caused by alanine
substitutions at these positions is MetR dependent
and because many of
these residues are situated near the

-DNA
binding residues, these
residues could be stabilizing the interaction
of

with DNA after
being positioned by MetR. Alternatively, these
residues could form a
protein-protein interaction surface for
contacting MetR. It has been
postulated that if indeed the

CTD
is involved in both nonspecific
DNA interactions and specific
protein-protein interactions, then
mutations in the residues involved
in the protein-protein interactions
should result in a stronger
phenotype than mutations in the DNA binding
residues (
1). Despite
the fact that the alanine substitution
experiments were performed
in the presence of a wild-type, chromosomal
copy of
rpoA, several
of these substitutions do indeed
exhibit stronger
metE phenotypes
than do substitutions in
the

-DNA binding residues (Fig.
2A).
In addition, a change from
isoleucine to alanine at residue 303,
which is buried within

but
behind residues E302, V306, and S309,
causes the most drastic reduction
in MetR-dependent activation
of
metE (Fig.
2A), presumably
by disrupting a MetR interaction
surface of

. In contrast, a change
from leucine to alanine at
270, which we propose disrupts the

-DNA
interaction by altering
helix 1 of the

CTD, has a more modest effect
on MetR-dependent
metE expression (Fig.
2A). Although we
favor a model where these
residues interact with MetR for activation,
we have not ruled
out the possibility that substitutions at these
residues may cause
a
metE-down phenotype due to some
indirect effect, e.g., altering
levels of MetR or expression of
homocysteine biosynthetic enzymes
in
vivo.
The other surface-exposed residues of the

CTD that
caused decreases in
metE activation when changed to
alanine, L289, S313,
and L314, do not localize to the complex face of

. However, these
residues, along with T285 (which causes nearly a
twofold decrease
in
metE activation in vivo when changed to
alanine [Fig.
2A]),
form a discrete patch on the face of the

CTD
opposite the complex
face (Fig.
5). These residues lie within the 20- by 10-Å surface
of the

CTD that has been shown to be important for
CRP activation
of the synthetic CC(

41.5) promoter and is proposed to
be the
surface used for an

CTD-CRP interaction (
36). The
CC(

41.5)
synthetic promoter has an activator binding site in
essentially
the same location (

41.5) as the activation site for MetR
on the
metE promoter (

42). Therefore, these residues could
define a
third MetR contact patch on the

CTD. The analogy between
metE and CC(

41.5) is not quite straightforward because of
the second,
upstream MetR binding site (site 1) at
metE that
is located in
the position thought to be contacted by the

CTD at
CC(

41.5);
however, since the rotational geometry of the proteins at
these
promoters is not known, this does not necessarily present a
steric
problem.
At the well-studied promoter CC(

41.5),

CTD-CRP and

CTD-DNA
interactions increase the binding of RNAP to the promoter but
have no
effect on the isomerization to open complex. CRP uses
additional
interactions with residues in the

NTD to facilitate
the
closed-to-open-complex isomerization (reviewed in reference
4a). Using CC(

41.5) as a model for
metE, we propose that RNAP
is recruited to
metE
through specific interactions between MetR
and residues T285, L289,
S313, and L314 of the

CTD. These residues
lie within the same 20- by
10-Å surface of

that contains the
residues important for the
recruitment of CRP to CC(

41.5) (
4a,
36). The MetR-RNAP
interaction may then be stabilized by

CTD-DNA
interactions involving
residues L262, R265, N268, C269, G296,
K298, and S299, which are
properly positioned to contact DNA upon
interaction between the

CTD
and MetR. The

CTD-DNA contacts made
at other promoters where the
activator binds overlapping the

35
sequence can be observed as an
extension of the footprint upstream
of the promoter in the presence of
wild-type RNAP but not with
RNAP carrying

subunits with CTD
deletions (for an example, see
reference
2),
suggesting that the

CTD reaches over the activator
to contact the
DNA. At
metE, however, an upstream extension of
the MetR
footprint is not observed with wild-type RNAP (
15a);
therefore, we favor a model where the

CTD interacts with DNA
within
the MetR footprint, probably by binding to a face of the
helix
different from that where MetR binds. This second, upstream
MetR
binding site at
metE may be the reason why we identified
additional residues within the complex face of

that are important
for MetR-dependent activation. Residues L260, T263, H276, P293,
E302,
V306, S309, N320, and P322 may also be used to stabilize
the MetR-RNAP
interaction. We did not identify any residues in
the

NTD that were
important for MetR-dependent activation; however,

NTD substitutions
did not, in general, cause strong decreases
in CRP-dependent activation
at CC(

41.5) (
31), so our initial
selection for
PCR-mutagenized
rpoA genes that decreased
metE
expression
may not have been sensitive enough to detect

NTD mutants.
Alternatively,
residues L260, T263, H276, P293, E302, V306, S309, N320,
and P322
of the

CTD may replace the

NTD-activator interaction
used at
CC(

41.5) to facilitate the closed-to-open-complex
isomerization.
Amino acids in the
CTD important for MetR activation of
metH.
In the initial selection for
mutations that alter
MetR-dependent activation, we were able to identify only one
substitution, N268D, which had a significant effect on metH
expression. Interestingly, we were able to detect a metH
phenotype only for the rpoA alleles tested in GS972, whereas
rpoA mutations that affected metE expression were
best detected in GS162 (Table 2). We speculate that a
metH-down phenotype is observed only in GS972 because GS162
does not produce enough MetR to fully activate metH, and a
metH-down phenotype is apparent only when there is
sufficient MetR available.
In the screen of the

CTD alanine substitution library, only

-DNA
interaction residues (R265, N268, K298, and S299) were
found to
significantly disrupt
metH activation if changed to alanine
(Fig.
2B). We confirmed the
metH phenotypes of R265 and N268
substitutions
in vitro as well as identifying another

-DNA
interaction residue,
G296, as important for
metH activation
(Fig.
3D). Therefore, five
of the residues previously identified as
important for

-DNA interactions
are also important for
metH activation: R265, N268, G296, K298,
and S299 (Fig.
5).
We were also able to show that mutations with
a slight phenotype in
vivo, e.g., L270H, could indeed cause a
significant phenotype in vitro
(Fig.
3D). Therefore, we consider
mutations that cause less than a
twofold down phenotype for
metH expression in vivo to be
potentially necessary for MetR activation
of
metH. By these
less stringent criteria, a number of other surface-exposed

CTD
residues, including D258, L262, T263, V264, C269, L290, P293,
N294,
L295, G296, and N320, also disrupt
metH activation if
changed
to alanine. Several of these residues have also previously been
shown to be important for activator-dependent transcription at
other
promoters: D258 is important for CRP, TyrR, and bacteriophage
Mu Mor
protein activation of
lac,
mtr, and
P
m, respectively (
1,
42,
55); V264 mutations can
suppress a positive control mutant
of OmpR to partially restore
activation at
ompF (
19); L290 is
necessary for P2
Ogr activation at P4 late promoters (
53); N294
is required
for activation at
katG by OxyR and UP element activation
of
rrnBp1 (
10,
44); and L295 mutations
disrupt UP element
activation of
rrnBp1
(
10).
All of the residues identified as crucial for
metH
activation are located within the complex face of

(Fig.
5). While
some
of the residues in this complex face are important for activation
of both
metE and
metH, others are important for
metH activation
only (Fig.
5). Substitutions in residues
D258, V264, L290, N294,
and L295 all affect
metH but not
metE expression. Since a number
of the
metH-specific residues have been shown to be important
for
activator-dependent expression in other systems, we propose
that
residues D258, T263, V264, L290, P293, N294, L295, and N320
contact
MetR for activation at
metH. Alternatively, D258 may
interact
with the
70 subunit, a role previously proposed
for this residue in CRP-dependent
activation at
lac
(
4a). Furthermore, we propose that the MetR-

CTD
interaction is stabilized by interactions between residues L262,
R265,
N268, C269, G296, K298, and S299 and
metH promoter
DNA.
The
metE and
metH promoters differ not only in
the number of MetR binding sites but also in the locations of the
activation
sites. However, a simple comparison of the locations of the
active
sites relative to the transcriptional start sites is problematic
because both S1 nuclease mapping (
47) and primer extension
(data
not shown) show that
metH has two transcription start
sites separated
by 3 intervening bp that appear to be used with equal
efficiency
in the absence and in the presence of MetR; thus, it is
likely
that both transcripts depend on the same Pribnow box. Because
of
the dual start sites at
metH, we use the 3'-most T base of
the Pribnow box, which is highly conserved in most promoters
(
13),
as a reference point to determine the relative
locations of the
MetR sites at the
metE and
metH
promoters. The center of the MetR
activation site (site 2) at
metE is 36 bp upstream of this reference
point, while the
center of the single MetR site at
metH is 52
bp upstream of
this point (Fig.
1); therefore, the MetR site at
metH is 16 bp, or one and one-half helical turns, further upstream
than the
metE promoter. This means that MetR binds to opposite
helical faces of DNA at these promoters. We propose that the
differences
in the

CTD residues that are important for MetR
activation result
from the differences in the locations of the MetR
activation sites
at
metE and
metH.
Differential orientation requirements for wild-type
CTD within
RNAP for activation at metE and metH.
Our
experiments with RNAP containing oriented
subunits indicate that
the CTD of either
subunit is capable of making the interactions
necessary for MetR-dependent activation at metE. This
interchangeability of the
CTD was also observed for UP element subsite recognition at rrnBp1 by Estrem et al.
(9). It has also been reported that the
CTD functions
interchangeably for CRP activation at lac and CC(
41.5)
(4a). In contrast, MetR activation at metH has a
more stringent requirement for an intact
CTD on the
-associated
I; the CTD of the
'-associated
II
substitutes very poorly for the
I CTD. The
metH promoter is similar to the lacP1 promoter in
that the activators bind well upstream of the
35 sequence in both cases; however, using the 3' T base of the Pribnow box as a reference, CRP binds to a site centered 54.5 bp upstream (6), while
MetR binding at metH is centered 52 bp upstream
(5). This means that CRP binds one-quarter of a helical turn
further upstream at lac than MetR binds at metH.
We speculate that the restriction on which of the two
CTDs is
capable of activation at metH is due to limits on the
rotational flexibility of the
CTD with respect to the rest of RNAP.
It has previously been shown that the
linker confers considerable
two-dimensional flexibility on the
CTD, allowing it to reach long
distances from the core promoter to interact with activator proteins,
as long as the helical phasing of the activator is maintained (11,
50, 52). Phasing experiments with FNR have shown that changes of
as little as 1 or 2 bp from a position favorable for FNR activation can
destroy FNR-dependent activation (52). From these results,
we speculate that the
CTD has considerable flexibility such that it
can stretch to reach distant activators but lacks a rotational
flexibility that would allow it to both reach for activators and wrap
around the DNA to contact activators that bind to a helical face of the
DNA different from that where RNAP binds. This restricted rotational
flexibility could also limit the mobility of each
CTD such that an
activator that binds "off to the side" of the DNA (relative to the
plane set by RNAP) would be able to contact one
CTD but the other
could not substitute if the critical
CTD was mutated or deleted.
This model would predict that other
CTD-dependent activators that bind "off to the side" may display an
-specificity, as was seen for MetR at metH. One example might be the OxyR-dependent
promoter katG, which has previously been shown to require
the
CTD (43). OxyR at katG binds 47 bp
upstream of the 3' T of the Pribnow box (45), meaning that
it also binds "off to the side" of the DNA but it binds to the side
opposite MetR at metH; therefore, we would predict that if
OxyR at katG does show an
CTD specificity, it may require
that the
CTD on the
'-associated
II be intact.
Furthermore, the limited rotational flexibility of the
CTD proposed
in this model would predict that
CTD-dependent activators that bind
to the opposite helical face of the DNA relative to RNAP would need to
bind in such a way that the activating region of the activator protein
would be wrapped around the DNA and would thus be accessible to the
CTD. Such a mechanism has the potential to lead to an
-specificity which could be examined in vitro by using oriented
RNAPs.
 |
ACKNOWLEDGMENTS |
We thank Robert Landick for purified
70 protein,
and Tamas Gaal, Richard Gourse, and Richard Ebright for plasmids.
This work was supported by a Carver Medical Research Initiative Grant.
P.S.F. is supported by a NIH Predoctoral Training Grant in
Biotechnology (GM08365) and The University of Iowa Center for Biocatalysis and Bioprocessing.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, 3-315A Bowen Science Building, The University of Iowa, Iowa City, IA 52242. Phone: (319) 335-7791. Fax: (319) 335-9006. E-mail: george-stauffer{at}uiowa.edu.
 |
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Journal of Bacteriology, October 2000, p. 5539-5550, Vol. 182, No. 19
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