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 His6-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
His6-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
His6-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, GS972TElac1, forms white colonies on
lactose tetrazolium agar when transformed with pREII. Transformants
of GS972TElac1 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 GS162TElac1 and GS972Hlac. 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, GS972TElac1, 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 GS972TElac1, and although
subcloning showed that the phenotype was associated with the
CTD, the subclones were also unstable in GS972TElac1.
Parallel subcloning of this mutant in GS162TElac1, 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, GS162TElac1 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 GS244TElac1, 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 GS972TElac1. The N268D
substitution decreased metE expression only threefold, compared to a ninefold decrease in GS162TElac1.
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 GS162Hlac, neither
mutation caused a decrease in metH-lacZ expression.
However, in GS972Hlac, 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 GS162TElac1 and GS972Hlac
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 GS162TElac1 were also tested in
GS244TElac1 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 GS244Hlac 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).
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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 GS162TElac1 lysogen,
GS1106TElac1, 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 GS972TElac1, 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 GS1106TElac1 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 L270HI-R45CII
heterodimers. The homodimeric L270H RNAP should be equivalent to the
purified His6-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
L270HI-R45CII 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 L270HI-R45CII 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 His6 tag nor a Strep tag
interferes with RNAP assembly or function, purified His6-
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 Ni2+ ion affinity of the His6-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
(CTDI-CTDII);
in the third and fourth RNAP species, the CTD was deleted from
either the -associated I
(CTDI-wt II) or the
'-associated II (wt
I-CTDII). 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,
CTDI-wt II and wt
I-CTDII, 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.
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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 CTDI-wt
II RNAPs, and 13 nM wt
I-CTDII and
CTDI-CTDII
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.
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The oriented RNAPs were also tested for their effects on
MetR-dependent expression of metH in vitro. Consistent with
previous results (15a), the
CTDI-CTDII
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.
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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.
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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
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Subunits in
MetR-Dependent Activation of metE and metH:
Important Residues in the C-Terminal Domain and Orientation
Requirements within RNA Polymerase
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Abstract
Introduction
