(Received for publication, July 5, 1995; and in revised form, October 18, 1995)
From the
The functional specificity was compared between two
factors,
(the major
at exponentially growing
phase) and
(the essential
at stationary
growth phase), of Escherichia coli RNA polymerase. The core
enzyme binding affinity of
was less than half the
level of
as measured by gel filtration column
chromatography or by titrating the concentration of
required for
the maximum transcription in the presence of a fixed amount of core
enzyme. In addition, the holoenzyme concentration required for the
maximum transcription of a fixed amount of templates was higher for E
than E
. The
transcription by E
was, however, enhanced
with the use of templates with low superhelical density, in good
agreement with the decrease in DNA superhelicity in the stationary
growth phase. We thus propose that the selective transcription of
stationary-specific genes by E
holoenzyme
requires either a specific reaction condition(s) or a specific
factor(s) such as template DNA with low superhelical density.
In Escherichia coli, the total number of RNA polymerase
core enzyme is fixed at a level characteristic of the rate of cell
growth, which ranges from 1,000 to 3,000 molecules per genome
equivalent of DNA(1, 2) . On the other hand, the total
number of genes on the E. coli genome is estimated to be about
4,000, which is in good agreement with the number estimated from the
DNA sequence (up to now, more than 60% has been sequenced). These
considerations raise a possibility that competition must take place
between promoters for binding a small number of RNA polymerase
molecules. Among about 4,000 genes on the E. coli genome,
about 1,000 genes are expressed at various levels in exponentially
growing cells under laboratory culture conditions, i.e. at 37
°C and with aeration(2, 3) . The rest of the genes
are considered to be expressed under various stress conditions that E. coli meets in nature (4, 5, 6, 7) . For instance, a set
of stress-response genes is expressed when cells stop growing at
stationary phase(6, 7) . Transcription of at least
some of these stationary phase-specific genes is catalyzed by RNA
polymerase holoenzyme containing (the rpoS gene product)(8, 9, 10, 11) .
In addition, the modification of core enzyme is considered to be
involved in stationary-specific transcription
regulation(12, 13) .
Promoters from the
stationary-specific genes, however, do not have a single consensus
sequence(10, 11, 14, 15) . The lack
of a consensus could indicate the involvement of a regulatory cascade,
in which some genes are directly transcribed by E but others are under the control of these gene products. However,
we found that the osmo-regulated genes, osmB and osmY, are transcribed preferentially by E
only in the presence of high
concentrations of potassium glutamate (or acetate) (16) . This
finding raises a possibility that each stationary-specific promoter
carries a specific sequence that is recognized by E
under a specific reaction condition and
suggests that the promoter sequences recognized by E
differ between gene groups with different
requirements. Our effort has since been focused to identify specific
conditions or factors required for transcription of each
stationary-specific gene by E
holoenzyme.
Along this line, we analyzed in this study the effect of DNA
superhelicity on the promoter recognition by E
holoenzyme. DNA superhelicity is known to change depending on the
cell growth conditions. For instance, nutrient downshift and stationary
growth phase cause a decrease in the DNA superhelical
density(17, 18) , while high osmolarity leads to an
increase in the superhelicity(19, 20) .
In this
report, we describe the comparison of two factors,
and
, in the following activities: (i) the
core binding activity, (ii) the promoter recognition activity, and
(iii) the effect of DNA conformation on the promoter recognition
patterns. The results show that the selectivity for stationary
phase-specific promoters by E
increases
concomitantly with the decrease in DNA superhelicity and that the
effects of decreased DNA superhelicity and high potassium glutamate
concentrations are additive in enhancing the selectivity for E
.
Plasmids
pBSOY containing osmY promoter and pBSLU containing lacUV5 promoter were constructed as follows. Plasmid
pBluescript II (Stratagene) was linearized with SacI and
blunt-ended using Klenow DNA polymerase; a 783-bp HincII-digested fragment containing rrnB terminator,
purified from pKK223-3 (Pharmacia), was ligated into pBluescript
II at the blunt-ended SacI site to yield plasmid pBST. A
261-bp HincII-EcoRV fragment containing osmY promoter, isolated from pDY.1.4(23) , was inserted into
pBST between HincII and EcoRV to yield pBSOY. On the
other hand, pBSLU was constructed after insertion of the 205-bp lacUV5 fragment from pKB252 (21) into pBST between HincII and EcoRI. These circular DNA templates
produced in vitro transcripts of 357 (lacUV5
promoter), 346 (osmY promoter), and 108 (primer RNA for ColE1)
nucleotides, respectively, in length. These template plasmids were
prepared from transformed DH5 cells using QIAGEN plasmid kit
(QIAGEN).
First, the affinity of the two subunits to core
enzyme was compared by directly measuring the holoenzyme formation. For
this purpose, we mixed a fixed amount of core enzyme and various
amounts of either
or
at 30
°C, and the mixtures were fractionated by gel filtration-HPLC on a
Superose 6 column. The
to core enzyme ratio was measured for the
peak fraction of RNA polymerase (Fig. 1). The core enzyme was
saturated with
at the input molar ratio between 2
and 3, while the saturation of the same amount of core enzyme with
required at least 2-fold more
protein than
. The observed difference might
be due to a difference in the activity of purified
subunits. To
test this possibility, the second-cycle assay was performed using the
unassembled
subunits recovered after the first cycle of binding
assay. The core binding patterns of both
subunits were
essentially the same with those of the first cycle experiments (data
not shown).
Figure 1:
saturation curve for
holoenzyme formation. Core enzyme (50 pmol) and various amounts of
or
subunit were mixed in 10
mM Tris-HCl (pH 7.8 at 4 °C), 10 mM MgCl
, 1 mM DTT, 0.1 mM EDTA, 0.2 M KCl, and 50% glycerol and incubated for 10 min at 30 °C
to form holoenzymes. The mixtures were fractionated by gel
filtration-HPLC using a Superose 6 (Pharmacia) column. Proteins were
eluted with 10 mM Tris-HCl (pH 7.8 at 4 °C), 200 mM NaCl, 0.1 mM DTT, 0.1 mM EDTA, and 5% glycerol.
Each fraction was analyzed by SDS-PAGE, and gels were stained with
Coomassie Brilliant Blue and scanned with an Ultroscan-XL laser
densitometer (LKB). The molar ratio of
,
`, and
was calculated for the peak fractions. The dotted lines represent the standard deviations of four or five independent
measurements.
Figure 2:
saturation curve for maximum
transcription. Core enzyme (1 pmol) and various amounts of
(A-C) or
(D) subunit were mixed as in Fig. 1. To the RNA
polymerase mixtures, one of the truncated DNA templates (0.1 pmol)
carrying fic (A), katE (B), lacUV5 (C), or alaS (D) promoter
was added and incubated for 30 min at 37 °C to form open complexes.
After addition of a substrate mixture containing
[
-
P]UTP as a labeled substrate, RNA
synthesis was carried out for 5 min at 37 °C. RNA was analyzed by
electrophoresis on 6% polyacrylamide gel in the presence of 8 M urea. Gels were analyzed with a BAS-2000 Bio-Imaging Analyzer
(Fuji). The transcription levels represent the average values of four
or five independent assays for each template and the standard
deviations. The 100% level corresponds to about 0.05 (about 0.5
molecule RNA per molecule DNA template), 0.03, 0.015, and 0.015 pmol
transcript for alaS, lacUV5, katE, and fic promoter, respectively.
Figure 3:
Effect of the holoenzyme concentration on
transcription. Truncated DNA templates (0.1 pmol each) carrying fic (A), katE (B), or lacUV5 (C) were transcribed in vitro by various amounts of
either E or E
holoenzyme under the standard single-round assay conditions.
Transcripts were analyzed by 8 M urea, 6% PAGE, and gels were
analyzed with a BAS-2000 Bio-Imaging Analyzer (Fuji). The maximum
levels of transcription for each template were set as 100% value,
measured using 4 pmol of E
for fic, 2 pmol of E
for katE, and 1-4 pmol of E
for lacUV5, respectively. The 100% value corresponds to about
0.015, 0.015, and 0.5 pmol transcript for fic, katE,
and lacUV5, respectively. The ratios of transcription levels
between two holoenzymes are plotted in the lower panels (fic (D), katE (E), and lacUV5 (F)).
The katE promoter was always transcribed better
with E than E
,
but the E
/E
activity ratio increased from 1.3 at 0.5 pmol (per 0.1 pmol
promoter; promoter/enzyme ratio of 5) to about 4 at the holoenzyme
concentration higher than 1 pmol. For all the promoters examined, the
transcription activity by E
increased at
high protein concentrations, supporting the notion that either the
affinity of
to core enzyme is weaker than that of
or the affinity of E
to
promoters is weaker than that of E
.
Figure 4:
Effect of the superhelical density of
template DNA on transcription. A set of circular DNA templates, pBSLU (A) and pBSOY (B) (0.1 pmol each), with various
superhelical densities was transcribed in vitro by
reconstituted E and E
holoenzymes under the standard single-round assay conditions.
Transcripts were subjected by 8 M urea, 4% PAGE, and the gel
was analyzed with a BAS-2000 Bio-Imaging Analyzer
(Fuji).
Figure 5: Effect of the superhelical density of template DNA on transcription. The in vitro transcription reaction was carried out as described in Fig. 4. Transcripts, lacUV5 RNA derived from pBSLU (A), osmY RNA derived from pBSOY (B), and RNA-I derived from the ColE1 origin promoter from both plasmids (C) were measured with a BAS-2000 Bio-Imaging Analyzer (Fuji). The maximum level of transcription for each template was set as 100% value. These data represent the averages of three independent experiments for lacUV5 and osmY and six independent experiments for RNA-I.
In
contrast, E required high levels of DNA
superhelicity for maximum activity. The optimum DNA superhelicity for
the maximum transcription of osmY promoter by E
was high (above 0.1), and upon decrease
in DNA superhelicity, E
becomes inactive in
transcription of the osmY promoter. Transcription of lacUV5 and RNA-I by E
was rather
insensitive to the change in DNA superhelicity (these promoters are
classified into a group of promoters that are recognized by both E
and E
holoenzymes(10, 15) ).
Figure 6:
Effect of cell growth phase on the
superhelical density of plasmid DNA. E. coli DH5 cells
carrying a plasmid, pBSOY, were grown in Luria-broth. At the times
indicated, the plasmid was isolated using QIAGEN plasmid kit, and the
superhelical density was determined by electrophoresis on 8% agarose
gels and in the presence of appropriate concentrations of ethidium
bromide as described under ``Materials and
Methods.''
Figure 7:
Promoter activity of plasmid DNA isolated
from exponentially growing and stationary phase cells. Low and high
superhelical density DNA templates (0.1 pmol each) carrying the osmY promoter were prepared from transformed DH5 cells at
4 and 24 h, respectively, after inoculation and transcribed in
vitro by various amounts of the reconstituted E
holoenzyme under the standard
single-round assay conditions. Transcripts were analyzed by 8 M urea, 4% PAGE, and gels were analyzed with a BAS-2000 Bio-Imaging
Analyzer (Fuji). The maximum level of transcription for the low
superhelical density template was set as 100%
value.
To confirm this prediction, we measured the rate of open
complex formation. The time course of open complex formation by E is roughly the same between the two
templates (the formation of open complexes was achieved within 15 min
of preincubation at 37 °C). However, the maximum level of open
complexes formed differed, indicating that the DNA superhelicity
difference does not affect the rate of open complex formation but
influences the promoter recognition activity by RNA polymerase (see
``Discussion'').
Figure 8:
Effect of potassium glutamate
concentrations on in vitro transcription of templates with
different superhelical densities. A and B, low and
high superhelical density DNA templates (0.1 pmol each) carrying lacUV5 promoter, were prepared from 4 and 24 h, respectively,
after inoculation and were transcribed in vitro by various
amounts of reconstituted E (A) and E
(B) holoenzymes under the
standard single-round assay conditions, except that 50 mM NaCl
was replaced with the indicated concentrations of potassium glutamate.
Transcripts were analyzed by 8 M urea, 4% PAGE, and gels were
analyzed with a BAS-2000 Bio-Imaging Analyzer (Fuji). The maximum level
of transcription for each template was set as 100% value. C and D, low and high superhelical density DNA templates
(0.1 pmol each) carrying the osmY promoter, prepared from 4
and 24 h, respectively, were transcribed in vitro by various
amounts of reconstituted E
(C) and E
(D) holoenzymes under the same
conditions as in panels A and B. Analyses of
transcripts were carried out as in panels A and B.
These data represent the averages of three independent
experiments.
The saturation experiments of in vitro transcription indicate that the affinity of
to
core enzyme is lower than that of
(see Fig. 2). Direct measurement of the core enzyme-bound
subunits by gel filtration-HPLC on Superose 6 column indeed showed that
the binding affinity of
to core enzyme is at least
2-fold weaker than that of
(see Fig. 1).
This was rather unexpected because the intracellular concentration of
is not higher than that of
even
after prolonged starvation in the stationary phase(26) .
Growth-coupled replacement of core enzyme-associated
subunit
from
to
may therefore require
an additional factor(s) or a yet unidentified condition(s). For
example, the modification of core enzyme in stationary phase cells (12, 13) may increase selective binding of
.
The single-round in vitro transcription assays also indicated that the level of E holoenzyme required for maximum
transcription of a fixed amount of template was higher than that of E
holoenzyme (see Fig. 2and Fig. 3). However, the maximum yields of transcription in the
presence of saturated amounts of enzyme were often lower than the
template levels and were different between the promoters. The
difference in abortive initiation between promoters provides a part of
the explanation as has been experimentally
demonstrated(27, 28) . In addition, the role of
sensitivity difference of various initiation complexes to heparin
cannot be excluded as a possible cause for the differing yields because
our preliminary gel retardation assay indicated that E
lacUV5 initiation complexes
were partly dissociated at 200 µg/ml heparin, while E
lacUV5 complexes stayed
unchanged. (
)
Another unexpected observation is that the
affinity of E holoenzyme to
-dependent promoters was rather weaker than that of E
to
-dependent
promoters at least under the standard transcription assay conditions
employed. Thus, in mixed transcription assays,
-dependent promoters are preferentially transcribed
at low enzyme concentrations, but upon the increase in enzyme
concentration, the
-dependent promoters become
predominantly transcribed. Again, this is apparently in conflict with
the intracellular concentrations of the two holoenzymes during the
transition from exponentially growing to stationary growth phase. Thus,
promoter-E
interaction may also be
influenced by a specific factor(s) and/or condition(s) present in
stationary phase E. coli cells. Previously, we found that
addition of high concentrations of potassium glutamate selectively
enhances transcription by E
, although E
activity is inhibited at the high salt
concentrations(16) . In addition, we found in this study that E
preferentially transcribed the promoters
on low superhelical density DNA (see Fig. 4and Fig. 5).
Moreover, the optimum superhelical density for maximum transcription by E
was almost the same as that of plasmids
prepared from stationary phase E. coli (see Fig. 6).
Preferential transcription of the low superhelical density templates by E
was due to the high affinity of E
binding to promoter on the low
superhelical density DNA (see Fig. 7).
The selective
transcription of osmY by E increases by adding high concentrations of potassium
glutamate(16) . The optimum potassium glutamate concentrations
for the maximum transcription of osmY on the stationary phase
plasmid template with low superhelical density by E
was 200-300 mM, where as
that on the DNA with high superhelical density prepared from log phase E. coli cells was above 300 mM (see Fig. 8).
Transcription in vivo of osmY in cells growing in
nutrient-rich media such as Luria-broth is observed only at the
stationary phase, whereas in cells growing in poor media such as
M9-glucose, osmY transcription occurs at both log and
stationary phases(23, 29) . In good agreement with
these observations, the superhelical density of plasmids prepared from
cells grown in M9-glucose is about half the density of plasmids
prepared from exponentially growing cells in Luria-broth.
These results altogether raise such a model of global regulation
that transcription by E
is enhanced with
the decrease in DNA superhelical density under starved conditions. In
addition, the increase in intracellular potassium glutamate
concentration may lead to enhanced transcription of at least
osmo-regulated genes. As an extension of this consideration, each of
the stationary phase-specific gene promoters may require a specific
transcription factor(s) or condition(s) for efficient transcription.
Such predictions are in good agreement with the observations that the
-dependent promoters do not carry a consensus
promoter
sequence(10, 11, 14, 15, 16) .
In addition to selective activation of E, a nucleoid-associated protein, H-NS (or
H1a), which accumulates in the stationary growth phase, represses
-dependent transcription in vitro and in vivo(30, 31, 32) . Likewise, Dps
accumulating in the stationary growth phase cells may play dual roles
in not only DNA protection but also repressing gene
transcription(33) . The differential repression by these DNA
binding proteins on transcription between E
and E
may also lead to apparently
selective transcription of
-dependent genes in the
stationary phase.