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Journal of Bacteriology, November 2001, p. 6315-6323, Vol. 183, No. 21
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.21.6315-6323.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Regulation of rRNA Transcription Correlates with
Nucleoside Triphosphate Sensing
Melanie M.
Barker and
Richard L.
Gourse*
Department of Bacteriology, University of
Wisconsin, Madison, Wisconsin 53706
Received 1 June 2001/Accepted 13 August 2001
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ABSTRACT |
We have previously shown that the activity of the
Escherichia coli rRNA promoter rrnB P1 in
vitro depends on the concentration of the initiating nucleotide, ATP,
and can respond to changes in ATP pools in vivo. We have proposed that
this nucleoside triphosphate (NTP) sensing might contribute to
regulation of rRNA transcription. To test this model, we have measured
the ATP requirements for transcription from 11 different
rrnB P1 core promoter mutants in vitro and compared them
with the regulatory responses of the same promoters in vivo. The seven
rrnB P1 variants that required much lower ATP
concentrations than the wild-type promoter for efficient transcription
in vitro were defective for response to growth rate changes in vivo
(growth rate-dependent regulation). In contrast, the four variants
requiring high ATP concentrations in vitro (like the wild-type
promoter) were regulated with the growth rate in vivo. We also observed
a correlation between NTP sensing in vitro and the response of the
promoters in vivo to deletion of the fis gene (an
example of homeostatic control), although this relationship was
not as tight as for growth rate-dependent regulation. We conclude that
the kinetic features responsible for the high ATP concentration
dependence of the rrnB P1 promoter in vitro are
responsible, at least in part, for the promoter's regulation in vivo,
consistent with the model in which rrnB P1 promoter
activity can be regulated by changes in NTP pools in vivo (or by
hypothetical factors that work at the same kinetic steps that make the
promoter sensitive to NTPs).
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INTRODUCTION |
In rapidly dividing
Escherichia coli, transcription from the seven rRNA operons
accounts for over half of all transcription in the cell (16, 26,
27, 37). rRNA operons are transcribed by two tandem promoters,
rrn P1 and rrn P2, with P1 being the predominant
promoter at medium to fast growth rates. Several features of
rrn P1 promoters contribute to their unusual strength. All seven P1 core promoters contain exact matches to the consensus 
10
hexamer (TATAAT) and close matches to the consensus 35 hexamer (TTGACA). All of the P1 promoters also derive much of their strength from UP elements, A+T-rich sequences (located from approximately 40
to 60 with respect to the transcription start site) that interact
with the 
subunit C-terminal domain of RNA polymerase (RNAP) and
activate transcription ~20- to 50-fold (28, 32, 52). In
addition, the transcription factor FIS binds to three to five sites
upstream of the UP element in each operon and activates transcription
approximately fivefold (32, 53).
While the rrn P1 promoters can be exceptionally strong, they
are also subject to regulatory controls that ensure that energy is not
wasted synthesizing excess translation machinery under less favorable
growth conditions (16, 26, 27, 37). There are multiple
ways in which these control systems have been assayed: responses of the
promoters to amino acid starvation (stringent control), to different
steady-state growth rates (growth rate-dependent control), and to
conditions that elicit a homeostatic response (see below).
Multiple molecular mechanisms likely underlie these regulatory
responses (27). For example, guanosine 5'-diphosphate,
3'-diphosphate (ppGpp) is responsible for inhibiting rRNA transcription
during the stringent response (12) but other regulatory
responses utilize different effectors. ppGpp is not essential for
growth rate-dependent or homeostatic regulation, since rrn
P1 promoter activity increases with growth rate and responds to at
least one type of homeostatic response in strains devoid of ppGpp
(5, 8, 22; see also reference 49).
rrn P1 promoters require much higher concentrations of the
initiating NTP (ATP or GTP) for maximal transcription in vitro than
most other promoters (7, 21), and rrnB P1 and
rrnD P1 promoter activities correlate with the levels of
their initiating NTPs (ATP and GTP, respectively) in strains with
altered NTP pools (21; D. A. Schneider and R. L. Gourse, unpublished data). These results indicate that rRNA
transcription can respond directly to variations in ATP and GTP
concentrations in vivo. We have proposed that NTP levels could serve as
indicators of the translational capacity of the cell and that
regulation by changing NTP concentrations, referred to as NTP sensing,
might be responsible for the increase in rrn P1 promoter
activity with growth rate. Furthermore, NTP sensing might also
contribute to the regulation of other promoters involved in the
synthesis of the translational machinery (49, 61).
However, since no methods are available with which to measure the
concentration of free NTPs in growing cells, and since measurements of
total NTP pools as a function of growth rate vary with the extraction
procedures used (21, 48; Schneider and Gourse, unpublished
data), it has been difficult to assess the role of NTP sensing in
growth rate-dependent regulation.
Homeostatic regulation (feedback control) of rRNA core promoter
activity keeps total rRNA synthesis relatively constant under conditions that might be expected to perturb it. Genetic alterations that would lead to under- or overproduction of ribosomes result in
compensating changes in rRNA core promoter activity. For example, rrn gene dose increases (by addition of rrn
operons on multicopy plasmids) result in corresponding decreases in
rrn P1 activity (29, 33) and rrn
gene dose decreases (by deletion of several rrn operons)
increase rrn P1 promoter activity (3, 15).
Likewise, decreases in upstream activation of rrn P1
promoters (by mutation of fis or rpoA) or
decreases in rRNA elongation (by mutation of genes for Nus factors)
increase rrn P1 promoter activity (50, 52, 53,
56). Consistent with the homeostatic regulation model,
rrn P1 promoter activity is stimulated by the protein
synthesis inhibitor chloramphenicol or spectinomycin in a futile
attempt to compensate for the resulting reduction in translational
capacity (57; Schneider and Gourse, unpublished data).
The molecular effector(s) responsible for homeostatic control is
unclear and could, in principle, be different in different experimental
situations. rrn P1 promoter activity decreases with an
increase in translationally competent ribosomes (but not with an
increase in translationally defective ribosomes), suggesting that the
feedback signal is either generated or consumed during protein
synthesis (14, 33, 62). Since translation is a major consumer of ATP and GTP, we have proposed that homeostatic control, like growth rate-dependent regulation, might be mediated by changes in
the concentration of available ATP and GTP (21).
Consistent with this hypothesis, treatment with protein synthesis
inhibitors not only increases rRNA transcription (see above) but also
increases ATP pools (Schneider and Gourse, unpublished data).
In this study, we explored whether two rrn P1 regulatory
responses are consistent with the predictions of the NTP sensing model.
That is, if NTP sensing were responsible for the changes in
rrn P1 promoter activity observed with increasing growth
rates or deletion of the fis gene, then it would be expected
that mutant rrn P1 promoters with altered responses to the
NTP concentration in vitro should display altered regulation in vivo.
We show that the ATP concentration dependences in vitro of 11 previously identified rrnB P1 promoter variants (17,
20, 35) correlate with their responses to growth rate and to
deletion of fis. These results are consistent with a simple
model in which growth rate-dependent regulation and, perhaps, feedback
control are mediated directly, at least in part, by changing
concentrations of the initiating nucleotide in vivo (or by a changing
parameter that works at the same kinetic steps that make the promoter
sensitive to NTPs).
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MATERIALS AND METHODS |
Promoter mutants, plasmids, strains, and bacteriophage.
The
plasmids and strains used in this study are listed in Table
1. Wild-type and variant rrnB
P1 promoters (66 to +9 with respect to the transcription start site)
were generated by PCR from plasmids containing the wild-type
rrnB P1 promoter or previously identified promoter variants
(17, 20, 35). The variants are described in Results, and
for consistency, the nomenclature used is the same as that employed
previously (17, 20, 35). DNA fragments were generated with
an EcoRI site at the upstream junction with the promoter
sequence and with a HindIII site at the downstream
junction, ligated into the transcription vector pRLG770
(53), and then cloned into bacteriophage 
to form
promoter-lacZ fusions in strain VH1000 (VH1000 = MG1655
pyrE+ lacI lacZ; courtesy of
V. J. Hernandez, State University of New York, Buffalo) as
previously described (50). Transductions of fis::kan-767 mutations were performed
with phage P1vir (44), using RJ1617 as the
donor strain (34).
ATP dependence assay.
E. coli RNAP
(E
70) was a generous gift from R. Landick and
was purified as previously described (11). Multiple-round
transcription reactions were performed essentially as previously
described (5, 6), using solution conditions in which the
rate constants affected by the initiating NTP are rate limiting.
Transcription was started by addition of 8 nM RNAP (~50% active) to
0.5 nM supercoiled plasmid in a mixture of 150 mM NaCl, 40 mM Tris-Cl
(pH 8.0), 10 mM MgCl2, 1 mM dithiothreitol, 0.1 µg of bovine serum albumin per µl, 200 µM CTP, 200 µM GTP, 10 µM UTP, 2 µCi of [-32P]UTP, and various
concentrations of ATP (5 µM to 2 mM) in 10-µl reactions at 30°C.
Transcription was terminated 20 min after RNAP addition with an equal
volume of formamide loading buffer. The reaction mixtures were
electrophoresed on 5% polyacrylamide-7 M urea gels, and the dried
gels were visualized and quantified by phosphorimaging (ImageQuant
Software; Molecular Dynamics). Fits to data points were made using
Sigmaplot (Jandel Scientific).
We measured the ATP dependence of the C-4T/A-3G promoter using a
concentration of CTP and GTP (20 µM) lower than those used
for the
other promoters (see above and Results). Control experiments
examining
the ATP dependences of the wild-type promoter and several
variants at
20 µM CTP and GTP showed that the [ATP]
1/2max
values
of the variant promoters relative to that of the wild type were
the same at both high and low CTP and GTP concentrations (data
not
shown).
Promoter activities at different growth rates.
Cells were
grown in Luria broth (LB) or in M9 minimal medium
(44) containing 0.4% glucose or glycerol, with or without
0.8% Casamino Acids (Difco) plus tryptophan (40 µg/ml). Liquid
cultures were inoculated to an A600 of
0.025 from fresh colonies. Cultures were grown at 30°C for about four
generations to an A600 of 0.35, harvested, and sonicated, and 
-galactosidase activity was measured (44). Cells can tolerate the extremely active
rrnB P1 promoters fused to lacZ using system I
(50), unlike the case for some other lacZ
fusion systems (58). However, the background activity in
system I fusions is relatively high. Background activity (60 to 120 Miller units, depending on the growth rate and genotype) was estimated
from the -galactosidase activities of a lysogen containing an M13
polylinker-lacZ fusion instead of a promoter-lacZ fusion. Appropriate background activities were subtracted from all of
the reported values, although it is possible that the insertion of the
promoter into the cloning site, in itself, eliminates the background.
In any case, the conclusions were not qualitatively different with or
without the subtraction of background activities.
Promoter activities in strains lacking the fis
gene.
-Galactosidase activities were measured as described
above for lysogens containing either a wild-type fis gene or
the fis::kan-767 insertion-deletion
(34). Cells were grown as described above in LB. We noted
that the activities of non-FIS-regulated promoter-lacZ fusions (e.g., PR and PhisG)
increased 2.6-fold in fis::kan strains for unknown reasons, as observed previously (53).
Therefore, only an increase in promoter activity significantly greater
than 2.6-fold (such as that observed for the wild-type rrnB
P1 promoter lacking FIS sites) was considered a specific response to
the absence of fis.
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RESULTS |
Choice of mutant rrnB P1 promoters.
The
rrnB P1 sequence endpoints required for regulation of
transcription initiation by growth rate in vivo are limited to the core
promoter (approximately 41 to +1 with respect to the transcription start site) (8). Furthermore, our previous mutagenesis
studies suggested that the sequence requirements for growth
rate-dependent regulation involve some of the sequence features that
are conserved among rrn P1 core promoters (17, 20,
35). We chose 11 previously identified rrnB P1 core
promoter mutants (17, 20, 35) for analysis of NTP sensing
in vitro (Fig. 1). These included a 1-bp substitution mutant (T-33A) with a consensus 35 hexamer, a 1-bp insertion mutant (Tins-23) with an increase in the length of the spacer
between the 10 and 35 hexamers to the E70
consensus (17 bp), and a double mutant that combines a 17-bp spacer
with a perfect 35 hexamer (T-33A/Ains-22), thus creating a consensus
core promoter. Three additional mutants with substitutions in positions
within the spacer were examined (C-17T, C-19T, and G-25T). To
investigate the region between the 10 hexamer and the transcription
start site, termed the discriminator because its high
G+C-content is characteristic of rRNA and most tRNA promoters (59), we examined a triple mutant and two single
substitutions that diminished this region's G+C content (CGC-5-7ATA,
C-5A, and C-4T) and a double mutant that altered the discriminator
sequence but preserved the G+C content (C-4T/A-3G). Lastly, we examined a mutant with a substitution at position 1 (C-1T); this position, which is conserved as a C in all seven rrn P1
promoters, was previously implicated in regulation by NTPs
(21).
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FIG. 1.
DNA sequences in the core promoter region of wild-type
and mutant rrnB P1 promoters. Numbers above the
wild-type sequence refer to promoter positions with respect to the
transcription initiation site. The 10 and 35 hexamers are
underlined and in bold in the wild-type promoter and underlined in
mutant promoters. Mutations are indicated in bold uppercase letters and
underlined. The rrnB P1 promoters analyzed extended from
66 to +9.
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In order to maximize basal promoter activity and thereby improve
measurement accuracy, both in vivo and in vitro, we examined
the 11 mutations in the context of promoters containing the UP
element. The
presence of an UP element has little or no effect
on the regulation of
wild-type
rrnB P1 by growth rate, feedback,
or NTP sensing
(
8,
21,
50,
52; M. M. Barker, T. Gaal,
W. Ross,
and R. L. Gourse, unpublished
data).
Several rrnB P1 variants display altered NTP sensing
in vitro.
To determine whether the variant promoters require
different initiating NTP concentrations than the wild-type
rrnB P1 promoter, we used an in vitro transcription assay in
which the concentration of ATP was varied from 5 to 2,000 µM while
the concentrations of CTP, GTP, and UTP were kept constant (Fig.
2). As shown previously (21), the wild-type rrnB P1 promoter requires a
relatively high ATP concentration for half-maximal transcription (under
the solution conditions employed, [ATP]1/2max
was ~250 µM). Seven mutant promoters (CGC-5-7ATA, T-33A/Ains-22,
Tins-23, T-33A, C-1T, C-5A, and C-4T) required at least threefold less
ATP than wild-type rrnB P1 for half-maximal transcription
([ATP]1/2max, <10 to 81 µM) (Fig. 2A). We
refer to the seven promoters with low initiating NTP concentration requirements as low-NTP mutants. Four variants (C-19T, C-4T/A-3G, C-17T, and G-25T) required ATP concentrations similar to or greater than that required by wild-type rrnB P1
([ATP]1/2max, ~205 to 430 µM ATP) (Fig.
2B). We refer to the promoters requiring high concentrations of the
initiating NTP in vitro (like wild-type rrnB P1) as high-NTP
mutants. For a summary of the
[ATP]1/2max values of the wild-type
and mutant promoters and the other regulatory properties of the
promoters (see below), see Fig. 4.
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FIG. 2.
Effect of ATP concentration on in vitro transcription of
wild-type and mutant rrnB P1 promoters. Transcription
was normalized to the highest level for each promoter. The wild-type
regression fit is in bold. (A) Mutants that required at least threefold
lower ATP concentrations for half-maximal transcription than wild-type
rrnB P1. (B) Mutants that required ATP concentrations
similar to or higher than that required by the wild type. Note the
different x-axis scale than in panel A.
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We emphasize the relative (and not absolute)
[ATP]
1/2max values of the different promoters,
since the absolute [ATP]
1/2max values
vary dramatically with the temperature, identity, and concentration
of
anions and cations in the reaction mixture and the superhelicity
of the
DNA template (
21) (data not shown). At relatively high
salt concentrations (e.g., 170 mM NaCl) and/or with nonsupercoiled
templates, millimolar ATP concentrations (i.e., in the range of
the
total ATP concentrations present in cells) are required for
maximal
activity of the wild-type
rrnB P1 promoter in vitro
(
21)
(data not shown). Most importantly,
rrnB
P1 activity responds
to both increases and decreases in the total ATP
concentration
in vivo (
21; Schneider and Gourse,
unpublished data). Therefore,
the free ATP concentration in cells is
likely not saturating for
initiation at this
promoter.
The behavior of the C-4T/A-3G variant was somewhat complicated. Unlike
that of the wild-type promoter, this mutant's transcription
start site
appeared to switch as the relative concentrations of
ATP, GTP, and CTP
were varied (data not shown). As a result, promoter
activity was
dependent on the ATP, GTP, and CTP concentrations,
depending on their
relative concentrations in vitro. Since the
start site switch occurred
when the ATP concentration was much
lower than the concentrations of
the other NTPs, and since this
condition is unlikely to occur in vivo,
we did not explore the
properties of the start site switch in more
detail. The ATP dependence
of C-4T/A-3G was measured at low GTP and CTP
concentrations in
order to keep the transcription start site the same
as for the
wild-type promoter (see Materials and Methods). Under these
conditions,
the C-4T/A-3G mutant required high levels of ATP
([ATP]
1/2max,
~300 µM), like wild-type
rrnB P1.
Low-NTP promoter mutants are defective for growth rate-dependent
regulation.
The NTP sensing model predicts that an rrnB
P1 variant requiring much less ATP than the wild type would be impaired
for growth rate regulation, since the free ATP concentration present in
cells would always be higher than that required for maximal
transcription (21). Conversely, the model predicts that an
rrnB P1 variant sensitive to changes in the concentration of
free ATP present in cells would be fully subject to growth rate regulation.
In order to test whether there is a correlation between the promoter
sequences required for NTP sensing and growth rate-dependent
regulation, single-copy
lacZ fusions to the wild-type and
mutant
promoters were constructed on the chromosome and their
activities
were measured as a function of the growth rate (Fig.
3A and B).
Since there are differences in
the mutant promoters' intrinsic
strengths, it is difficult to
visualize the effects of the mutations
on regulation without
normalization for promoter activity. Figure
3C and D illustrate the
growth rate dependence of the wild-type
and mutant promoters after
their activities were scaled to the
same value at the lowest growth
rate.
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FIG. 3.
Growth rate-dependent control (GRDC) of wild-type and
mutant rrnB P1 promoters. Each panel includes data
points from three independent experiments for each promoter. The
wild-type regression fit is in bold. Note that all of the panels have
different ordinate scales. The top panels display the actual
-galactosidase activities versus the growth rate (doublings per
hour). The bottom panels display the promoter activities normalized at
the lowest growth rate in order to facilitate visualization of defects
in regulation (see text). (A and C) Mutants that are defective for
growth rate-dependent regulation. (B and D) Mutants with activities
that increase with growth rate similarly to that of the wild type.
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The responses of the
rrnB P1 variants to changes in growth
rate fell into two major classes. Whereas the wild-type promoter's
activity increased about 10-fold over growth rates ranging from
0.3 to
1.5 doublings per h, seven mutants (CGC-5-7ATA, T-33A/Ains-22,
Tins-23, T-33A, C-1T, C-5A, and C-4T) increased only 1.4- to 4-fold
with increasing growth rates, much less than wild-type
rrnB
P1.
In contrast, four mutants (C-19T, C-4T/A-3G, C-17T, and G-25T)
increased 7- to 20-fold with increasing growth rates, to extents
similar to or greater than that of the wild-type
promoter.
The average fold increases from the lowest to the highest growth rate
for the wild-type and variant promoters are summarized
in Fig.
4B. Although this type of representation
tends to exaggerate
small differences in the primary data (e.g., the
differences in
NTP sensing of the four high-NTP mutants appears to be
much more
significant in Fig.
4A than in Fig.
2B), it is clear from
comparison
of Fig.
4A and B that the seven mutants most defective for
growth
rate-dependent regulation are the same mutants most defective
for NTP sensing. In contrast, the four mutants that are growth
rate
regulated most like the wild-type promoter are the ones most
sensitive
to the same range of ATP concentrations as the wild
type. Thus,
although there may be some minor differences in the
rank order of the
extents of growth rate-dependent regulation
and NTP sensing (see
Discussion), we concluded that the
cis-acting
sequences in
rrnB P1 needed for NTP sensing correlate well with
the
sequences required for growth rate-dependent regulation.
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FIG. 4.
Summary of regulation of wild-type (wt) and mutant
rrnB P1 promoters. Promoter mutants are listed in the
same order for each panel, from the promoter with the lowest
[ATP]1/2max to that with the highest. The bars for the
wild-type promoter are black, those for the seven low-NTP, growth rate
regulation-defective mutants are light gray, and those for the four
high-NTP, growth rate regulated mutants are dark gray. The bars for the
promoter mutants in panel C are not separated into classes because
distinctions are somewhat ambiguous in this assay. (A) The mean
[ATP]1/2max values were determined from at least two
independent experiments. Variation was less than 20%, except for C-19T
(27%). (B) Average fold increase with growth rate was calculated for
the wild type and mutants (except C-17T) by dividing the
-galactosidase activity in LB (µ = ~1.4) by the activity in
M9 glycerol (µ = ~0.33). The averages and standard deviations
(less than 14%) were calculated for three independent experiments. The
activity of the C-17T promoter is too close to the background in M9
glycerol for accurate assessment, so its fold increase was estimated by
dividing the activity in LB by the activity in M9 glucose (µ = 0.58). C-17T increased approximately twofold more than the wild-type
promoter in this growth rate range (standard deviation = 26%).
Therefore, its fold increase with growth rate may be an underestimate.
(C) Feedback derepression in fis mutant strains.
fis/wild-type promoter activity ratio is reported as in
Table 2.
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Low-NTP promoter mutants are defective for feedback derepression in
fis::kan strains.
We next
examined the relationship between a promoter's NTP concentration
requirement and its response in one of the several assays that have
been used to estimate feedback (homeostatic) control. In strains
with the fis gene deleted, rRNA core promoter activity
increases to partially compensate for the loss of activation (50,
53). If the mechanism responsible for this feedback regulation is NTP sensing, then the promoter sequences important for a response to
the loss of FIS-dependent activation should correlate with those
required for NTP sensing. Therefore, we compared the activities of the
wild-type and mutant rrnB P1 promoters in wild-type strains or strains with fis deleted.
As observed previously (
50,
53), for reasons that are
unclear (see Materials and Methods), the activities of all
promoter-
lacZ fusions increased in
fis::
kan mutants. However, as also
observed
previously, the activity of the wild-type
rrnB P1
promoter lacking
FIS sites increased in a
fis::
kan strain 1.5-fold more than the
activities of control promoters (3.8-fold versus 2.6-fold; Table
2). In general, the mutants that were
defective for NTP sensing
in vitro responded to the loss of
fis more like the control promoters
than like wild-type
rrnB P1; i.e., the promoters requiring the
highest ATP
concentrations (C-17T, G-25T, and C-4T/A-3G) increased
the most in
response to the loss of
fis, the promoters requiring
the
lowest ATP concentrations (CGC-5-7ATA, T-33A/Ains-22, Tins-23,
and
T-33A) increased the least in response to the loss of
fis,
and the promoters with intermediate ATP concentration requirements
(C-1T, C-5A, C-4T, and C-19T) had intermediate responses to the
loss of
fis. However, because of differences in rank order and
the
relatively modest regulatory effect observed in this assay,
we have not
grouped the promoters into the same classes as for
NTP sensing in vitro
and for growth rate-dependent control in
vivo (Table
2; Fig.
4C).
Nevertheless, we conclude that the promoters'
response to the loss of
the
fis gene correlates qualitatively
with their response to
changing NTP concentrations. Potential
explanations for the less than
perfect correlation between NTP
sensing in vitro and the response to
the loss of
fis in vivo are
discussed further below.
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DISCUSSION |
Regulation by NTPs in vitro correlates with growth rate-dependent
regulation and feedback derepression in
fis::kan strains in vivo.
By analysis of the regulation of rrnB P1 promoter variants,
we have shown that the concentration of the initiating NTP required in
vitro for efficient transcription of a particular promoter correlates
well with its susceptibility to growth rate-dependent regulation and
more qualitatively with feedback control in vivo. As a result, our
primary conclusion is that the kinetic features responsible for the
dependence of rrn P1 promoters on high initiating NTP
concentrations in vitro make these promoters sensitive, at least in
part, to changes in growth rate and loss of fis in vivo. We
noted that different mutations likely alter the topology and salt
concentration dependences of each promoter to somewhat different extents. Thus, it is all the more remarkable that the ATP concentration dependences of the different promoters under any single condition in
vitro correlate as well as they do with regulation in vivo.
Models of rrn P1 regulation by growth rate and
feedback.
There are two general models to explain the mechanism of
rRNA regulation that are consistent with the observed correlations. The
simplest model is that rrn P1 promoters monitor the levels of free initiating NTP pools to coordinate rRNA synthesis rates with
protein synthesis rates. This model is attractive because it explains
how rRNA synthesis can be regulated, at least in part, by the overall
biosynthetic energy capacity of the cell as a function of growth rate,
and it also suggests the identity of one of the feedback signals
generated by transient under- or overproduction of ribosomes.
We cannot exclude a more complicated alternative model in which one or
more unidentified regulatory signals are elicited in
response to growth
rate changes and loss of the
fis gene and these
signals work
on the same kinetic steps that make the promoter
dependent on high
concentrations of the initiating NTP for maximal
transcription. We have
used mutants with altered purine or pyrimidine
metabolism to
demonstrate that
rrn P1 promoter activity changes
when total
NTP pools are altered (
21; Schneider and Gourse,
unpublished data). Although these studies suggest that NTP
concentrations
are not saturating for
rrn P1 promoters in
vivo, we cannot rule
out the possibility that free NTP pools do not
change in vivo
as a function of growth rate or in
fis::
kan strains. In any case,
because
free NTP pools are likely subsaturating, hypothetical
factors that
affect the same kinetic steps that make the promoter
sensitive to NTPs
could theoretically alter the kinetics of initiation
in
vivo.
In lieu of an assay capable of measuring free NTP pools, our finding
that there is a good correlation between NTP requirements
in vitro and
regulation of rRNA transcription in vivo supports
the NTP sensing
model. We note that it is well established that
NTP concentration
changes can affect the transcription of pyrimidine
biosynthetic operons
(although by using mechanisms different from
that proposed for the
regulation of rRNA transcription) (
13,
30,
39,
43).
Kinetic basis for regulation by NTPs.
During the multistep
process of transcription initiation (51), RNAP first binds
the promoter to form a short-lived closed complex. The closed complex
can then isomerize through at least one intermediate to form an open
complex in which the DNA in the 10 hexamer and start site region is
locally unwound. rrnB P1 and other similarly regulated rRNA
and tRNA promoters form open complexes with half-lives of a few seconds
or minutes under solution conditions in which most other promoters form
open complexes with half-lives of several hours (5, 25, 31, 38,
40, 41, 49).
The unusually short-lived open complex formed by
rrn P1
promoters makes them susceptible to regulatory molecules that alter
open-complex occupancy. For example, ppGpp shortens the half-life
of
all RNAP-promoter open complexes in vitro, independently of
whether or
not the promoter is regulated by ppGpp in vivo (
5).
However, ppGpp only inhibits transcription from those promoters,
like
rrnB P1, that form intrinsically short-lived complexes
(i.e.,
where the open-complex half-life is rate limiting for
transcription
initiation), explaining its specificity in vivo
(
5). We propose
that the effect of initiating NTP
concentration on rRNA promoters
can be explained in a similar way:
although all promoters bind
NTPs to initiate transcription, the free
NTP concentrations present
in vivo are limiting for those promoters
whose open complexes
are exceptionally short-lived (
21).
We have characterized the kinetic properties of the
rrnB P1
promoter mutants described here (M. M. Barker, T. Gaal, W. Ross,
and R. L. Gourse, unpublished data). The seven low-NTP promoters
form complexes 4-fold to more than 30-fold longer-lived than wild-type
rrnB P1. In contrast, the high-NTP mutants form open
complexes
with short lifetimes, similar to the wild-type promoter.
Therefore,
we propose that for a promoter to be susceptible to
regulation
by the initiating NTP concentration, the rate of
open-complex
collapse must be competitive with the time required for
initiation.
In theory, there could be promoters with short half-lives
and
with very fast forward rate constants. Therefore, we speculate
that
NTP sensing might have a second requirement: the forward
rate constants
cannot be so fast that the open complex accumulates
even in the absence
of the initiating
NTP.
The promoter sequence determinants for regulation are complex.
The sequence determinants for regulation are multipartite and involve
several of the sequence and structural characteristics common to
rrn P1 promoters, including nonconsensus 35 hexamers, 16-bp spacers, G+C-rich discriminators, and a C at position 1 (see also references 5, 19, 31, 36, 37, 47, 49,
and 63). Other promoter positions also could be important for regulation, since our screens were not exhaustive, and promoter context is likely to play an important role. In addition, transcription initiation occurs farther from the 10 hexamer in rrn P1
promoters than in most promoters and it is possible that atypical
positioning of the start site plays a role in regulation.
As part of an extensive survey of a large number of promoter mutants,
we previously characterized the growth rate-dependent
regulation (but
not the feedback regulation or NTP concentration
requirements) of the
promoter mutants described here (
8,
17,
35). For 3 of the
11 mutants characterized here (C-5A, C-17T,
and G-25T), the degree of
growth rate-dependent regulation differs
somewhat from that reported
previously. For technical reasons,
we believe that the results reported
here are more
accurate.
Multiple mechanisms contribute to regulation of rRNA
transcription.
Some of the same kinetic features that make rRNA
promoters sensitive to changing NTP concentrations also make rRNA
promoters sensitive to other potential regulators (4-6).
Although strains lacking ppGpp or FIS retain relatively normal growth
rate-dependent regulation of rRNA transcription, these regulators could
work in conjunction with the effects of NTPs. Moreover, since the level of negative supercoiling and the concentrations of monovalent and
divalent cations also affect the rrnB P1 open-complex
lifetime in vitro (25, 41; M.M.B. and R.L.G., unpublished
data), systematic changes in superhelicity or osmolarity in vivo (if
they occur) have the potential to work in conjunction with NTPs and
ppGpp during changes in growth rate or in fis deletion
strains. However, unlike the case with NTPs, which are consumed by
protein synthesis and thus could serve as indicators of the ribosome
level, it is unclear why or how supercoiling and osmolarity should
change under these conditions.
We think it unlikely that another recently proposed regulatory
mechanism, changes in the free RNAP concentration (
42), is
a major contributor to the control of rRNA transcription in vivo.
rrn P1 promoters require lower concentrations of RNAP for
transcription
in vitro than most other promoters (
4), and
rRNA transcription
does not respond in vivo to changes in the RNAP
concentration
that affect transcription from mRNA promoters (
4,
9,
46).
Close examination of Fig.
4 reveals that the rank order and extent of
the mutant promoters' responses to NTP concentration
changes in vitro,
to growth rate changes, and/or to deletion of
fis in vivo
are not always exactly the same. These disparities
could result simply
from compounding of errors associated with
comparison of ratios or from
the inability of in vitro assays
to exactly duplicate conditions in
cells. Most notably, while
the most NTP-responsive mutants responded
most to the loss of
fis and the least NTP-responsive mutants
responded least to the
loss of
fis, the relatively narrow
window of the regulatory response
in this assay makes quantitative
assessment difficult. We also
emphasize that the changes in
rrnB P1 promoter activity associated
with changes in growth
rate or loss of the
fis gene could be mediated
only in part
by variations in NTPs, and quantitative discrepancies
in the
correlations between the in vitro NTP-sensing behavior
of the promoters
and their in vivo regulatory responses could
reflect the participation
of other components in the
system.
Squires and coworkers have suggested that feedback elicited by an
increased or decreased rRNA gene dose might be mediated
by a mechanism
distinct from that responsible for growth rate-dependent
regulation and
NTP sensing (
60). They studied four
rrnB P1
variants
from our collection (T-33A, Ains-22, C-1T/C-15G, and C-1T)
(
17)
and found an incomplete correlation between the
cis-acting sequences
required for growth rate-dependent
regulation and those required
for a response to gene dose changes.
Given the complexity intrinsic
to
rrn P1 promoters and their
regulation, it seems entirely reasonable
that different molecular
mechanisms might operate under different
conditions eliciting a
feedback response. For example, part of
the compensating increase in
rRNA transcription that occurs in
fis deletion strains
probably is an increase in
rrn P2 promoter
activity
(42a).
Many regulators of rRNA transcription influence the levels or
activities of other contributors. For example, deletions of
the
fis or
hns gene (H-NS is a histone-like protein
that appears
to negatively regulate
rrnB P1 under some
conditions [
1,
55])
can lead to changes in DNA
supercoiling (
45,
54). FIS not
only recruits RNAP to
rrn P1 promoters (
10) but also reduces
the ATP
concentration required for
rrn P1 transcription
(
6).
Induction of ppGpp synthesis reduces ATP and GTP
pools (
23,
24), and the ATP/ADP ratio may regulate
supercoiling levels
in vivo (
18). Thus, the output from
all of the regulatory mechanisms
affecting rRNA transcription may be
more than the simple sum of
the
inputs.
| |
ACKNOWLEDGMENTS |
We thank Tamas Gaal, Wilma Ross, David Schneider, and other
members of our laboratory for insightful discussions and/or comments on
the manuscript. We also thank Bob Landick for the generous gift of RNAP
and Christine Hirvonen for cloning the lysogen containing the M13
polylinker-lacZ fusion.
This work was supported by NIH grant GM37048 to R.L.G. and a fellowship
from Pfizer Biotechnology to M.M.B.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Bacteriology, University of Wisconsin, 1550 Linden Dr., Madison, WI
53706. Phone: (608) 262-9813. Fax: (608) 262-9865. E-mail:
rgourse{at}bact.wisc.edu.
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Journal of Bacteriology, November 2001, p. 6315-6323, Vol. 183, No. 21
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.21.6315-6323.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
