From the
We have previously shown that the accumulation of 20 tRNA
species in Escherichia coli is individually regulated as a
function of cellular growth rate. We have also reported that the growth
rate regulation of some but not all tRNA species is dependent on the
activity of the factor for inversion stimulation (FIS). In present
work, we studied the growth rate regulation of the serine- and
threonine-accepting tRNA families. We show that the levels of
tRNA
The tRNA multigene family of Escherichia coli consists
of 79 genes that are distributed in 41 transcription units, encoding 46
structurally distinct tRNA species (41 different
anticodons)(1) . Some tRNA genes are located in the spacer
regions within the seven rRNA operons. Others are located in operons
coding only for tRNA or are cotranscribed with protein coding
sequences. The large number and disparate structure of tRNA operons
make the study of tRNA gene expression complicated. In addition to the
growth rate control, two mechanisms that actively regulate the
accumulation of stable RNA, tRNA and rRNA, have been described: the
ppGpp
The involvement of ppGpp
in repressing the synthesis of stable RNA in response to amino acid or
energy source starvation is well documented (2). It has also been
suggested that ppGpp is involved in the growth rate regulation of
stable RNA synthesis since the level of ppGpp is inversely correlated
with the expression level of stable RNA at different growth
rates(4) . Furthermore, bacteria that are deficient in degrading
ppGpp grow at slower growth rates and have reduced rRNA transcription
rates compared with wild type bacteria(5, 6) . However,
deletion of the genes that encode the two ppGpp synthetases results in
a bacterium (relA, spoT) that, albeit with a slower
growth rate, regulates stable RNA synthesis in a normal growth
rate-dependent manner(7) . This suggests either that ppGpp is
not involved in the growth rate control or alternatively that some
other regulatory mechanisms take over in the absence of ppGpp. One such
mode of regulation has been described as ribosomal feedback inhibition
(8). This phenomenon was observed in cells that overproduced rRNA from
plasmids(8, 9) . The expression of plasmid-borne rRNA
genes resulted in reduced expression of the chromosomal rRNA and tRNA
genes. The ribosomal feedback inhibition mechanism depends on the
formation of functionally initiated ribosomes(10) , but the
details of this mechanism are not known.
Some stable RNA operons
have been shown to be activated by FIS, in vitro or in
vivo(11, 12, 13) . FIS-dependent activation
is regulated by de novo synthesis of FIS protein, where the fis gene is both auto-regulated and subject to stringent
control(14, 15) . Upon dilution of stationary phase
cultures in fresh medium or after a nutritional up-shift, the level of
the FIS protein increases rapidly and reaches a peak after
approximately one generation(15, 16) . The data suggest
that FIS is involved in accelerating bacterial growth rate under those
conditions.
In order to understand how the composition of the tRNA
pools is regulated in E. coli, we have determined the growth
rate-dependent level of individual tRNA species in strains where the fis gene is deleted. We have previously studied the tRNA
species belonging to the arginine, leucine, and methionine isoacceptor
families(17) . In this report, we have studied the growth rate
regulation of the serine and threonine-accepting tRNA species in E.
coli strain W1485 and in a isogenic fis bacteria. Five
tRNA species show a strong dependence on a functional FIS protein for
their accumulation. Here, the levels of tRNA are reduced as much as
5-fold in fis bacteria. Expression of the other three tRNA
species are identical in wild type and fis bacteria. These
data provide support for the suggestion that the FIS protein is
involved in the high expression level of many tRNA genes at fast
bacterial growth rates.
Inactivating the fis gene does not change the growth
rate-dependent expression of the rRNA operons(12, 17) .
We have therefore used 16 S rRNA as an internal standard and expressed
our data as the ratio of tRNA to 16 S rRNA. The target specificity of
the eight oligonucleotides used has been tested, showing that the
probes are tRNA-specific.
Accumulation of tRNA
The impetus for this study is to illustrate further the
involvement of FIS in the growth rate-dependent regulation of
individual tRNA species in E. coli. In a previous study, we
found that the growth rate-dependent expression of arginine,
methionine, and leucine-accepting tRNA species can be divided into four
groups pertaining to the effect of deleting the fis gene(17) . The accumulation rate of one group of tRNA
species decreases in the absence of FIS. A second group of tRNA species
have been shown to increase their accumulation rate in the absence of
FIS. The accumulation rate of a third group of tRNA species is growth
rate-regulated but unaltered in fis cells. Thus, the
regulatory response to the absence of FIS for these tRNA species is
similar to the response found for rRNA(12) . A fourth group
contain tRNA species with a weak or undetectable increase in
accumulation rate at faster growth rates, thus seeming to be under no
growth rate regulation. The accumulation of these tRNA species is also
unaffected by the absence of FIS. In the present study, we show that
tRNA species within the serine and threonine acceptor families fall
into two of these groups (Fig. 1).
The expression
levels of the three tRNA genes encoding tRNA
In
the absence of the upstream activating sequences, transcription from
the thrU(tufB) promoter is drastically increased in fis cells compared to wild type bacteria(11, 16) . The thrU(tufB) promoter lacking an upstream activating sequence
cannot bind FIS in vitro(11, 32) , indicating
that the lowered expression from the upstream activating sequence-less
construct in wild type cells is not due to an inhibitory binding of FIS
to the promoter region. Such a compensatory mechanism is also noted for
the rrnB operon ending at position +1(12) , which
argues against the involvement of any downstream elements. We suggest
that this compensation is caused by the ribosomal feedback system.
Evidence exists that ppGpp is not involved in the ribosomal feedback
inhibition. Gaal and Gourse (7) have shown that the growth
rate-dependent regulation of the rrnB operon is unaffected in
cells incapable of producing ppGpp. This seems not to be caused by an
increased FIS-dependent activation of the rrnB operon since a
minipromoter construct without any FIS binding sites is still growth
rate-regulated in ppGpp-less bacteria (33). Furthermore, deletions of
one or several rRNA operons result in increased expression of the
remaining rRNA operons. This is not accompanied by a decrease in ppGpp
levels, suggesting that the ribosomal feedback inhibition is uncoupled
from the ppGpp-dependent regulatory mechanism(34) . However, it
should be noted that the findings above have been questioned by
Hernandez and Bremer(35) , who have found that the rrnB promoter is not growth rate-regulated in cells lacking ppGpp. It
is also noticeable that both the ppGpp-dependent repression of stable
RNA synthesis and the ribosomal feedback mechanism have been shown to
depend on the promoter region only(36, 37, 38) .
We suggest that the combination of FIS-dependent activation and
inhibition due to the ribosomal feedback system determines the growth
rate-dependent regulation of the tRNA genes. We suggest that the four
groups of tRNA species that we find correspond to tRNA species located
in operons regulated by 1) FIS, 2) the ribosomal feedback system, 3)
both FIS and the ribosomal feedback system, and 4) none of these
mechanisms. Genes regulated by FIS alone should have a growth
rate-dependent regulation that disappears or is severely reduced in fis cells. Transfer RNA
We have
demonstrated a strong dependence on a functional FIS protein for the
gene expression of several tRNA species under steady state growth rates
of bacteria. We find that for tRNA genes that are dependent on FIS for
their up-regulation, the requirement for FIS is most significant under
rich growth conditions. Furthermore, the collected data of the 20 tRNA
species studied so far suggest that the abundance of bulk tRNA is
reduced in fis cells at fast growth rates. The limited supply
of translating tRNA species and the unbalance in the tRNA pool in fis cells may partly explain the marked differences in the
growth rates between wild type and fis bacteria in rich media.
The gift of the MC1000-fis767 clone by R.
Johnson and the construction of W1485-fis767 strain by D.
Hughes are gratefully acknowledged.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, tRNA
,
tRNA
, tRNA
, and
tRNA
are reduced in fis cells as the
growth rate increases. The accumulation of these tRNA species is
reduced 2-5-fold at the fastest bacterial growth rate. The
strongest effect is observed for the two minor tRNA species;
tRNA
and tRNA
. In
contrast, we find that the accumulation of
tRNA
, tRNA
, and
tRNA
is similar in wild type and fis bacteria. The data presented provide further evidence for the
suggestion that FIS is a stimulating factor that is involved, directly
or indirectly, in the high expression level of some tRNA genes at fast
bacterial growth rates.
(
)-dependent stringent control and the more
recently described factor for inversion stimulation (FIS)-dependent
activation (for reviews see Refs. 2 and 3).
Chemicals and
Enzymes
[-
P]ATP, polynucleotide
kinase, and the Hybond-N
membranes were purchased from
Amersham International (UK). Formamide, phenol, and the components used
for the electrophoresis were of ultrapure grade and purchased from
International Biotechnologies Inc.
Bacterial Strains
The bacterial strains used for
RNA determinations were E. coli W1485 (18) and
W1485-fis767 (fis cells)(17, 19) .
Cells were grown in M9 medium(20) , supplemented with vitamin B1
(0.01 mM), FeCl (0.03 mM), CaCl
(0.1 mM), MgSO
(1 mM), and 0.4% of
acetate, succinate, or glucose as the carbon source. In addition,
cultures were grown in rich medium containing 20 amino acids, purines,
and pyrimidines as described by Neidhardt et al. (21) and LB medium containing 0.4% glucose (20).
RNA Isolation
Cells were grown at 37°C with
vigorous shaking in a water bath. The cultures were grown in a steady
state phase for at least 10 generations as described by Emilsson and
Kurland (22) and were harvested on ice at an A of 0.8. The cells were lysed, and the RNA was extracted as
described by Emilsson and Kurland(22) .
Electrophoretic Fractionation and Northern
Hybridization
Approximately 25 µg of the crude RNA extract
was fractionated on horizontal 1.2% agarose gels in TAE buffer (40
mM Tris acetate, pH 7.5, and 1 mM EDTA) and then
transferred to Amersham Hybond-N hybridization
filters. The filters were hybridized with specific probes for 16 S rRNA
and one tRNA species as described previously(17, 22) .
The labeled bands were identified by autoradiography, excised, and
measured in a liquid scintillation counter for radioactive
quantitation. The films were also analyzed by densitometric scanning
counter. The tRNA-specific oligodeoxyribonucleotide probes were
complementary to the 3`-half of the molecules, i.e. the
variable loop, anticodon stem, and anticodon loop sequences of the
tRNAs studied (5` to 3`): Ser1: AACCCTTTCGGGTCGCCGGTTTTC; Ser2:
GTAGAGTTGCCCCTACTCCGGT; Ser3: CCCCGGATGCAGCTTTTGACC; Ser5:
ATACGTTGCCGTATACACAC; Thr1: CTGGGGACCCCACCCCT; Thr2: CCTACGACCTTCGCATT;
Thr3: CTGCCGACCTCACCCTT; Thr4: CTGGTGACCTACTGATT. The 16 S
rRNA-specific oligodeoxyribonucleotide probe is an 18-mer complementary
to the conserved region between positions 562 and 579(23) .
(
)In this study, we
find two modes of tRNA regulation. Some tRNA species such as
tRNA
have reduced expression in fis cells, whereas others like tRNA
are
unaffected.
The Serine Acceptor tRNA Family
Four tRNA species belong
to the serine-accepting tRNA family in E. coli,
tRNA, tRNA
,
tRNA
, and tRNA
.
According to predictions based on the unmodified anticodon,
tRNA
translates the minor UC(A/G) codons.
Transfer RNA
translates the two major serine
codons UC(U/C), while the two rare serine-accepting tRNAs,
tRNA
and tRNA
,
translate the minor serine codons UCG and AG(U/C), respectively.
, encoded by a single
gene in the serT operon(1) , is moderately decoupled
from the accumulation of 16 S rRNA in both wild type and fis bacteria (Fig. 2), while the accumulation of the rare
tRNA
, encoded for by the serU operon(1) , is severely reduced in the absence of FIS (, Fig. 1, and Fig. 2). Here,
tRNA
accumulation is coupled to the
production of 16 S rRNA in wild type cells at various growth rates,
while in fis cells the level of tRNA
drops 5-fold under the fastest growth condition.
Figure 2:
Northern blot analysis of the four tRNA
subspecies belonging to the serine acceptor tRNA family. The ratio of
tRNA to 16 S rRNA in wild type bacteria at the slowest growth rate is
taken as 1. The bars indicate standard errors calculated from
six independent experiments. The intracellular concentrations of the
tRNA species at the growth rate of 0.5 doublings/h are marked in parentheses with the value for the wild type strain first. A, tRNA (6.3 µM, 4.8
µM); B, tRNA
(1.6
µM, 1.4 µM); C,
tRNA
(1.9 µM, 1.8
µM); D, tRNA
(10
µM, 9.5 µM).
Figure 1:
Autoradiographs of
Northern blot hybridizations of RNA fractionated in 1.2% agarose gels.
The hybridizations were performed with probes specific for 16 S rRNA as
well as for tRNA (A) and
tRNA
(B). The RNA preparations were
from strain W1485 (lanes1-5) and strain
W1485-fis767 (lanes 6-10). The bacteria were
grown in M9 + acetate (lanes1 and 6),
M9 + succinate (lanes2 and 7), M9
+ glucose (lanes3 and 8), complete
medium (lanes4 and 9), and LB medium +
glucose (lanes5 and 10).
The
accumulation of another rare serine acceptor tRNA,
tRNA, is found to be coupled to the
production of rRNA in wild type bacteria. In contrast, accumulation of
tRNA
is decoupled from the production of 16 S
rRNA in fis cells although not as drastically as we observe
for tRNA
. Transfer RNA
is cotranscribed with four copies of the tRNA
gene in the serV operon(1) , and its regulatory
response is similar to the previously described
tRNA
. Finally, the 16 S rRNA-normalized
levels of the abundant tRNA
, transcribed by
two separate operons at positions 20` and 23`, are constant at
different growth rates in both wild type and fis cells (, Fig. 1, and Fig. 2). In conclusion,
tRNA
and tRNA
belong to the group of tRNA species that depend on FIS for their
growth rate regulation. The tRNA
and
tRNA
species behave as rRNA; they are growth
rate-regulated, but the accumulation of the gene products is unaffected
by deletion of the fis gene.
The Threonine Acceptor tRNA Family
The threonine
isoacceptor tRNA family in E. coli, consists of four species
with three unique anticodons(1) . Predictions based on the
unmodified anticodon suggest that tRNA and
tRNA
, with identical anticodons, translate
the major threonine codons, AC(U/C), whereas tRNA
and tRNA
translate the minor ACG and
AC(A/G) codons, respectively. The gene for threonine isoacceptor 1 is
located in the ribosomal RNA operon, rrnD (1). As expected,
the accumulation of tRNA
is coupled to the
accumulation of 16 S rRNA in both wild type and fis cells (see Fig. 3). In contrast, regulation of
tRNA
, expressed from the thrW operon(1) , is strongly affected by the deletion of the fis gene ( and Fig. 3). There is already a
35% reduction in the abundance of this tRNA at the slowest growth rate
in fis bacteria, and the level decreases additionally 3-fold
at the fastest growth rate.
Figure 3:
Northern blot analysis of the four tRNA
subspecies belonging to the threonine-accepting family. The ratio of
tRNA to 16 S rRNA in wild type bacteria at the slowest growth rate is
taken as 1. The bars indicate standard errors calculated from
six independent experiments. The intracellular concentrations of the
tRNA species at the growth rate of 0.5 doublings/h are marked in parentheses with the value for the wild type strain first. A, tRNA (1.6 µM, 1.5
µM); B, tRNA
(2.5
µM, 1.6 µM); C,
tRNA
(3.8 µM, 3.5
µM); D, tRNA
(3.5
µM, 2.9 µM).
Two threonine-accepting tRNA species,
tRNA and tRNA
, are
expressed from the thr(U)tufB operon(1) . These tRNA
genes are located in the same operon and are therefore expected to be
expressed in a similar way at different growth rates. Indeed, in wild
type cells the accumulation of both tRNA species is coupled to the
accumulation of 16 S rRNA at various growth rates (Fig. 3). In fis cells growing in rich medium, the expression of these two
tRNA genes is reduced 2-fold (), which suggests that FIS is
required for their high expression level. These results are consistent
with the data of Nilsson et al.(11, 16) .
However, the present data contradicts the finding by Lazarus and
Travers(25) , showing that the absence of FIS does not affect
the expression level of the thr(U)tufB operon. Summarizing the
regulation of the threonine-accepting tRNA family,
tRNA
, tRNA
, and
tRNA
depend on FIS for their growth rate
regulation. The tRNA
is growth
rate-regulated, but the accumulation of the gene products is unaffected
by deletion of the fis gene.
tRNA Species with Reduced Accumulation Rates in fis
Cells
The growth rate-dependent accumulation of the
tRNA, tRNA
,
tRNA
, tRNA
, and
tRNA
species is reduced in fis cells, suggesting that they are, directly or indirectly, activated
by FIS and that the expression cannot be fully compensated for by
relaxation of the ribosomal feedback system (see below). In fact, the
accumulation of the rare tRNA species tRNA
and tRNA
is severely affected by the
deletion of the fis gene, with a 5-fold lower accumulation
rate under the fastest growth condition. We have previously shown that
also the rare tRNA species tRNA
and
tRNA
are affected by lack of FIS(17) .
Thus, in our studies, all rare tRNA species that are growth
rate-regulated depend on FIS for this regulation. It has been suggested
that two of these rare tRNA species are required for mechanisms other
than translation. The cell division mutation ftsM1 has been
mapped to the tRNA
gene(26) , and the
tRNA
gene complements a temperature-sensitive dnaY mutation. Since this mutation affects the polymerization
phase of DNA replication, tRNA
has been
implicated in the replication process(27) . It is shown that fis cells are defective in DNA replication and cell division
control(28, 29) . FIS binds to the origin of replication (oriC), and it has been suggested that the lack of FIS bound
to oriC in fis cells causes the replication and cell
division defects. However, a recent study shows that FIS does not
stimulate the replication process in vitro(30) . An
alternative explanation is that the fis cell defects are
caused by reduced levels of tRNA
and
tRNA
. An altered cell morphology, presumably
due to the cell division and replication defects, is only seen at fast
growth rates of fis cells, when the levels of
tRNA
and tRNA
are
most dramatically reduced.
(
)
,
tRNA
, and tRNA
are
also reduced when the fis gene is deleted but to a lesser
degree than the levels of tRNA
and
tRNA
. The tRNA
and
tRNA
genes are located in the thrU(tufB) operon. This operon was the first to be shown to be regulated by
FIS(11) . Using an operon fusion where the tufB gene
was replaced with the gene for galactokinase, it was shown that
approximately half of the growth rate-dependent increase in the
expression of galactokinase depended on the presence of the FIS-binding
sequence upstream of the promoter(16) . More recently, Lazarus
and Travers (25) have reported that the wild type thrU(tufB) promoter is not FIS-dependent. They ascribed the previous result
to a cryptic down-mutation in the promoter region. This discussion is
made more complicated by the finding of Vind et
al.(31) , showing that the expression of reporter genes
from strong promoters on multicopy plasmids is not linear in relation
to mRNA levels. We demonstrate that fast growing fis cells
contain half the amount of the threonine-accepting tRNA species that is
under the control of the thrU(tufB) promoter compared with
wild type cells. Our data are in good agreement with previously
reported data (16) but in clear contrast to the data of Lazarus and
Travers(25) .
tRNA Species with Unaltered Accumulation Rates in fis
Cells
The accumulation rates of the Ser1, Ser5, and Thr1 tRNA
species increase at faster growth rates. However, the expression level
of these RNA species is not reduced by the deletion of the fis gene. These and other tRNA species whose growth rate-dependent
regulation responds only mildly to the absence of FIS must therefore be
regulated by other means. There is evidence suggesting that at least
one mechanism by which these tRNA species are regulated is identical to
the ribosomal feedback system, as discussed below.
Regulation of tRNA Expression
It is clear that the
accumulation rate of individual tRNA species respond differently to the
absence of FIS. What causes these differences? It is a strong
correlation between the accumulation rates of different tRNA species
within one operon, both in the presence and absence of
FIS(17, 22, 24) . We also observed this pattern
in the present study. Here, the Thr3 and Thr4 tRNA species, which genes
are located in the thrU(tufB) operon, are coupled to the
accumulation of 16 S rRNA in wild type cells while their accumulation
is decoupled from 16 S rRNA production in fis cells. Also,
Ser3 and Arg2 tRNA species are regulated identically in both strains
(see Ref. 17 for Arg2 tRNA). Finally, the expression of the Thr1 tRNA
gene, located in rrnD together with rRNA genes, is coupled to
the expression of rRNA in wild type and fis bacteria at
various growth rates. Our data suggest that tRNA accumulation is
regulated predominantly at the transcription initiation level.
and
tRNA
belong to this group. Genes regulated by
the ribosomal feedback system only should have an increased expression
in fis cells. We did not find tRNA species in the threonine
and serine families with this regulatory pattern, but we have
previously shown that tRNA
,
tRNA
, and tRNA
belong to this group(17) . If the gene is regulated by
both mechanisms, a relaxation of the ribosomal feedback system should
compensate for the absence of FIS, as is the case for the expression
from rrnB(12) . In this investigation,
tRNA
, tRNA
, and
tRNA
illustrate this group. The last group of
tRNA species have poor or no growth rate regulation, suggesting that
they are neither regulated by FIS nor by the ribosomal feedback system.
None of the threonine or serine tRNA species belong to this group, but
previous results show that tRNA
,
tRNA
, tRNA
, and
tRNA
have a poor growth rate regulation and
the that expression of these tRNA species is unaffected by the absence
of FIS(17) . Intermediates between the groups are also found.
Several tRNA species such as tRNA
,
tRNA
, tRNA
, and
tRNA
have slightly reduced expression in the
absence of FIS ( Fig. 3and Ref. 17), as expected if the ribosomal
feedback system only partly compensates for the lack of FIS.
Table: Comparison of individual tRNA levels
accumulated in wild type and fis bacteria, grown in various medium
compositions
bfrensen, M. A., Rasmussen, M. D., and Pedersen, S. (1993) J. Mol. Biol.231, 678-688
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