(Received for publication, March 9, 1995; and in revised form, May 12, 1995)
From the
It has been shown previously that two different mRNA chains (lacZ and infB) are elongated at a rate of
When Escherichia coli is exposed to amino acid
starvation or other nutritional down-shifts, it undergoes the so-called
stringent response, meaning that the synthesis of stable RNA (i.e. tRNA and rRNA) stops abruptly, while bulk mRNA synthesis continues
(Cashel and Rudd, 1987), although at reduced rate (Svitil et
al., 1993; Set al., 1994). Very early in
the stringent response, the unusual nucleotide guanosine
3`,5`-bis(diphosphate), also called ppGpp, accumulates at high
concentrations in the cells and is responsible, somehow, for the
selective stop of stable RNA synthesis, since relA mutants
unable to synthesize high concentrations of ppGpp fail to undergo the
stringent response (Cashel and Gallant, 1969; Cashel and Rudd, 1987;
Hazeltine and Block, 1973; Pedersen et al., 1973; Stent and
Brenner, 1961). Despite much research, the mechanism by which
accumulation of ppGpp leads to down-regulation of tRNA and rRNA
synthesis is unknown. Bremer and collaborators (Baracchini and Bremer,
1988; Bremer and Dennis, 1987; Tedin and Bremer, 1992) have suggested
that the binding of ppGpp to RNA polymerase leads to partitioning of
the enzyme in two forms with different promoter specificity and that
the RNA polymerase bound with ppGpp is unable to initiate transcription
at stable RNA promoters. On the other hand, Jensen and Pedersen(1990)
have proposed that ppGpp inhibits stable RNA synthesis indirectly by
inhibiting RNA chain elongation and thereby sequestering RNA polymerase
in the elongation phase. Thus, ppGpp was proposed to lower the
concentration of free RNA polymerase and reduce the initiation of
transcription, primarily from the ribosomal RNA promoters because these
strong promoters were considered to be difficult to saturate with RNA
polymerase because of unusually high k To test the effects of ppGpp on RNA chain
growth in vivo, we previously measured the rate of
transcription elongation on different genes under different growth
conditions of E. coli. We found that the rate of transcription
elongation on two different mRNA genes (i.e.lacZ and infB) was about 40 nt( One difference between the employed mRNA genes and
the ribosomal operons is that the latter contain antiterminator
sequences similar to the nut sites of bacteriophage The experiments reported in this paper
were aimed at clarifying the mechanistic basis for the different
responses to ppGpp pools variations between transcription complexes on
ribosomal RNA operons and mRNA genes. First, we have studied the rate
of transcription elongation in a
There are at least two straightforward explanations for the
faster transcription elongation on rRNA genes compared to mRNA genes.
1) The rRNA sequences may lack ppGpp-dependent pause sites, presumed to
make RNA polymerase elongate mRNA chains more slowly when the ppGpp
pool increases. 2) The antiterminator boxes and the putative factors,
which may bind to RNA polymerase at the boxes, might make the
polymerase resistant to ppGpp-dependent (and ppGpp-independent)
pausing, thereby increasing the over-all transcription elongation rate.
To distinguish between these non-excluding alternatives, we have
studied the effects on transcription elongation of removing the
antiterminator boxes from a ribosomal operon and of inserting an
antiterminator boxA sequence in the early transcribed regions of infB and lacZ. Measurements of the rates of
transcription elongation were made according to a previously published
procedure (Vogel et al., 1992) using the plasmids described in Fig. 1. Thus, the time lag between induction of a plasmid-borne, lacI repressor-controlled promoter and detection of specific
hybridization to a probe complementary to the 3` end of the transcript
was measured and taken to represent the transcription time of the gene.
The promoter on these plasmids is a modified T7 A1 promoter, the
P
Figure 1:
Schematic presentation of the employed
plasmids. Genes are represented by openbars. P/O, promoter/operator site of P
Figure 2:
Autoradiograms of dot blots from induction
experiments using MAS90 harboring pUV25. Induction was performed during
growth on glucose minimal medium. Top, steady state growth; bottom, isoleucine starvation induced by addition of valine
(0.4 mg/ml) 6 min prior to induction of transcription with IPTG (1
mM). The first sample is the uninduced background, followed by
samples are taken out every 10 s from 20 to 120
s.
Figure 3:
Induction
of lacZ mRNA synthesis in MAS90 harboring either pUV12 (A and C) or pUV16 (B and D). MAS90 was
grown on glucose minimal medium. At time = 0, IPTG was added at
1 mM. Transcription was either induced during steady state
growth (A and B) or 6 min after the onset of
isoleucine starvation induced by addition of 0.4 mg/ml valine (C and D). RNA was extracted at the indicated times after
induction and used for dot blot hybridization with the 3`-lac probe (see Fig. 1). The amount of hybridization was
quantified on an Instant Imager (Canberra-Packard) and cpm was plotted versus sampling time. All the experiments were performed at
least two times.
Figure 4:
Induction of the 5`-lacZ mRNA in
MAS90 carrying either pUV12 (A) or pUV16 (B) using
the 5`-lacZ probe (see Fig. 1). Transcription was
either induced during steady state growth (filledcircles) or 6 min after the onset of isoleucine
starvation induced by addition of 0.4 mg/ml valine (opencircles). The RNA samples were the same as those used for Fig. 3, but the 5`-lac probe (Fig. 1) was used
for quantitative hybridization. The experiments with pUV12 were made
twice.
Figure 5:
Induction of infB mRNA synthesis
in MAS90 harboring pUV25. Transcription is induced either during steady
state growth (A) or 6 min after the onset of isoleucine
starvation induced by the addition of 0.4 mg/ml valine (B).
The experimental procedures were the same as those for Fig. 3.
Fig. 6shows the transcription
kinetics from lacZ (pUV12) and from the antiterminated lacZ (pUV16) in wild type MG1655 (CF1648) and in the
Figure 6:
Induction experiments using CF1648 relA
Figure 7:
Square root plot of the increment in
As shown in Fig. 8(panels
A-C), removal of the antitermination boxes led to a
successive increase in the transcription time during steady state
growth, i.e. from 45 s for pUV17, to 55 s for pUV22 and 70 s
for pUV26. These numbers correspond to transcription elongation rates
of 80 nt/s in the presence of both antiterminator boxes (pUV17), a
transcription elongation rate of 65 nt/s when only the antiterminator
in the spacer region is present (pUV22) and 50 nt/s without any of the
two antiterminators (pUV26), but the transcription elongation rate even
without any antiterminator boxes is still significantly higher than we
observe for the mRNAs, i.e.
Figure 8:
Induction of truncated rRNA in MAS90
carrying either pUV17 (A and D), pUV22 (B and E), or pUV26 (C and F). MAS90 was
grown on glucose minimal medium. At time = 0, IPTG was added at
1 mM. Transcription was either induced during steady state
growth (A-C) or 6 min after the onset of isoleucine
starvation induced by addition of 0.4 mg/ml valine (D-F). RNA was extracted at the indicated times after
induction and used for dot blot hybridization with the cat probe. The amount of hybridization was quantified on in an Instant
Imager (Canberra-Packard) and cpm was plotted versus sampling
time. All the experiments were performed twice. The data in panelD were taken from (Vogel and Jensen, 1994a) and are
incorporated in the figure for clarity
reasons.
Our results demonstrate that the boxA sequence, in addition
to the well established role in antitermination, also functions as a
determinant of the rate of transcription elongation. Moreover, it
appears that the presence of an antiterminator boxA early after the
promoter makes transcription elongation insensitive to inhibition by
ppGpp. The results of all transcription experiments are summarized in Table 1. It is clear that boxA divides the data in two groups on
the basis of elongation rates, since the RNA chains that carry the boxA
sequence are elongated at high rates (62-82 nt/s), while the RNAs
that do not have boxA are elongated more slowly (19-52 nt/s). It
is also clear that the elongation rate of mRNAs, devoid of the boxA
sequence, responds strongly (up to 2.6-fold) to changes in the ppGpp
pool, while the elongation rate of the corresponding mRNAs carrying
boxA only changes by 25-30% in response to similar changes to the
ppGpp pool. These observations indicate that the boxA sequence, or the
protein factors that associate with RNA polymerases at boxA, make RNA
polymerase more resistant to ppGpp inhibition.
In this context, the
results obtained from transcription studies of a segment of rRNA are
relevant. These data also support the notion that boxA is a major
determinant of the RNA chain elongation rate, since transcription of
rRNA in the absence of antitermination sequences occurred with an
elongation rate of 50 or 80 nt/s in the presence of antiterminator
sequences. However, even after deletion of the antiterminator boxes,
the elongation rate of the truncated rrnX operon DNA was still
higher than observed for the mRNAs lacZ and infB, and
it did not respond significantly to ppGpp accumulation during the
stringent response. That observation may indicate that the rDNA segment
on these plasmids is devoid of ppGpp-dependent pause sites for
transcription. The absence of such transcriptional pauses would also
explain why the elongation rate of rRNA lacking antiterminator
sequences (50 nt/s) is equal to the elongation rate of the lacZ mRNA in the ppGpp Under
conditions of isoleucine starvation, we see about 80-90%
transcription polarity in lacZ, and that polarity is
completely suppressed in the boxA-lacZ fusion. Also, the data
in Table 1indicate the existence of a correlation between a low
rate of transcription elongation and a high degree of transcriptional
polarity, since the stringent response induced 80-90% polarity in
the lacZ gene (see text), Somehow, we find it
puzzling that rRNA transcription elongation is insensitive to ppGpp,
since the down-regulation of ribosomal RNA synthesis is known as a
primary target for ppGpp action during the stringent response (Cashel
and Rudd, 1987). On the other hand, since the high concentration of
ppGpp accumulating under the stringent response inhibits mRNA
elongation, ppGpp will serve to minimize decoupling of the ribosomes
from the RNA polymerase, and thereby reduce the level of premature
transcription termination, under growth conditions that give rise to
suboptimal ribosome function. Furthermore, high concentrations of ppGpp
lead to reduction of the total mRNA pool; thus, indirectly, ppGpp
enhances the accuracy of translation during shortage of substrates for
the elongating ribosomes (Svitil et al., 1993; Set al., 1993, 1994). It appears as if the `naked` RNA
polymerase is designed to elongate slowly enough to allow ribosomes to
catch up and to be inhibited to ppGpp, which is a signal for ribosome
starvation. However, the RNA polymerase clearly has the capacity to
elongate RNA chains much faster, as evidenced by the high elongation
rate of ribosomal RNA. The function of the high transcription
elongation rate of rRNA may be to make the polymerase less prone to
terminate transcription, as proposed for Rho-dependent termination on
the basis of analysis of RNA polymerase mutants (Jin et al.,
1992), and to reduce the number of RNA polymerases needed for rRNA
transcription and thus for total transcription. To conclude, the
antiterminator boxes seem to have three functions: 1) they prevent
polarity, 2) they increase the rate of transcription elongation, and 3)
they confer ppGpp insensitivity to the RNA polymerase. In turn, these
observations indicate that these functions of the RNA polymerase
(polarity, elongation rate, and ppGpp sensitivity) are intimately
coupled with each other.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
40
nucleotides (nt)/s during steady state growth on minimal medium and
that the rate of mRNA chain elongation is inhibited by ppGpp in
vivo. On the other hand, it was found that a truncated ribosomal
RNA chain was elongated at a rate of
80 nt/s, independent of
growth condition (Vogel, U., and Jensen, K. F. (1994) J. Biol.
Chem. 269, 16236-16241). We reasoned that the different
transcriptional behavior of mRNA genes and rRNA operons might be caused
by the antiterminator sequences present in the rRNA operons. To test
this possibility, we have (a) inserted the minimal
antiterminator boxA sequence between the promoter and the lacZ and infB genes and (b) deleted the
antiterminator sequences from the rRNA transcription unit and measured
transcription elongation rates in vivo on the resulting hybrid
genes. We found that insertion of boxA in front of the coding region of lacZ increased the transcription elongation rate from 42 nt/s
to 69 nt/s during steady state growth and that it eliminated the
ppGpp-dependent decrease in the transcription elongation rate during
the stringent response. On the other hand, deletion of the
antiterminator sequences from the rRNA operon resulted in a reduced
transcription elongation rate, but the elongation rate was still
insensitive to changes in the ppGpp pool. These results are consistent
with the hypothesis that the antiterminator boxA is a primary
determinant of the rate of transcription elongation rate.
/K
values (Jensen
and Pedersen, 1990).
)/s during exponential growth
on a glucose-salt minimal medium and that it varied slightly with the
growth rate (Vogel and Jensen, 1994b). The elongation rate of these
mRNAs was approximately halved during the stringent response to
isoleucine starvation, when ppGpp accumulated in vast amounts, and it
increased to
50 nt/s during the relaxed response to isoleucine
starvation, when the basal ppGpp pool decreased to almost undetectable
levels (Vogel and Jensen, 1994a; Vogel et al., 1992). These
results indicated strongly that mRNA chain elongation is subject to
ppGpp inhibition in vivo, since the changes in the elongation
rates occurred within the first minute after the onset of isoleucine
starvation (Vogel et al., 1992). However, the elongation rate
of a truncated ribosomal RNA was close to 80 nt/s, both during steady
state growth (in agreement with some previous estimates for intact rRNA
(Condon et al., 1993; Molin, 1976) and during amino acid
starvation of relA and rel
strains,
indicating that ppGpp does not inhibit ribosomal RNA chain elongation in vivo.
(Aksoy et al., 1984; Li et al., 1984) Each ribosomal
operon contains two antiterminator sequences, one sequence is placed
just after the P2 promoter and the other is positioned in the spacer
region between the 16 and 23 S RNA cistrons (Berg et al.,
1989). The antiterminators contain three recognizable RNA sequence
motifs, namely boxA, boxB, and boxC, but the boxA sequence was shown to
be both necessary and sufficient for proper antitermination in the
ribosomal operons in vivo and in vitro (Berg et
al., 1989; Squires et al., 1993). They are expected to
cause binding of different protein factors to the transcription
elongation complex (Squires et al., 1993) as shown for the nut sites of bacteriophage
(Horwitz et al.,
1989), but the binding of such factors to the ribosomal antitermination
sites has not been shown.
relA
spoT strain (i.e. a ppGpp
mutant devoid of ppGpp) to observe
the effects of basal level ppGpp pools without disturbing the growth.
Second, we have studied the effects on transcription elongation of
inserting the antiterminator boxA in front of the coding regions of infB and lacZ transcripts and of removal of the
antiterminator boxes from a ribosomal operon. The results show
conclusively that the antiterminator boxA is a major determinant of
both transcription elongation rate and of resistance to ppGpp pool
changes in vivo.
Strains and Plasmids
Unless stated otherwise,
the background strain used for these experiments is MAS90, i.e.E. coli K-12 thi pro-lac
relA2/F`lacI
lacZ::Tn5 proAB
,
which has been described previously (Vogel and Jensen, 1994a). CF1693
(MG1655
relA251
spoT207) and CF1648 (MG1655) (Xiao et al., 1991) were gifts from V. J. Hernandez (National
Institutes of Health, Bethesda, MD). The F` proAB
lacI
lacZ
M15
Tn10 from SØ6645 (MC1061, pyrD::Km
/F` proAB
lacI
lacZ
M15
Tn10) (own collection), which confers tetracycline resistance, was
mated into the two strains selecting tetracycline resistance and
pyrimidine prototrophy. The transconjugants were subsequently
transformed with either pUV12 or pUV16 selecting for ampicillin
resistance. The construction of the plasmids pUV12, pUV14, and pUV17 is
described elsewhere (Vogel and Jensen, 1994a).
Construction of pUV22
An EcoRI-EcoRI fragment from pUV17 containing the
minimal boxA and 2122-bp rDNA was exchanged with an EcoRI-EcoRI fragment from pKK3535 (Brosius et
al., 1981) containing the same 2122-bp sequence and 30 additional
bp in the 5` end of the rDNA fragment, but not containing the minimal
boxA.Construction of pUV26
The antitermination
sequences in the spacer region of the rDNA in pUV22 were removed by PCR
amplifying a segment of rDNA on either side of the antiterminator
sequences, incorporating a BamHI site into the primers. The 5`
PCR fragment had an XmaI site in the rDNA sequence in the 5`
end and a BamHI site in the 3` end. The 3` PCR fragment had a BamHI site in the 5` end and a BssHII site in the
rDNA sequence at the 3` end. The two PCR fragments were digested with
the appropriate restriction enzymes and ligated into XmaI-BssHII-digested pUV22 in a three-fragment
cloning resulting in deletion of 84-bp rDNA and insertion of 4 bp from
the artificial BamHI site.Construction of pUV16
pUHE23-2 is a cloning vector
containing the synthetic, IPTG-inducible promoter P,
which is a derivative of the T7 A1 promoter. The promoter is fused with
two lac operator sites, followed by a polylinker, a terminator
structure, a cat gene, and the ribosomal T1 terminator. The
plasmid confers ampicillin resistance (H. Bujard, University of
Heidelberg, Germany). A DNA duplex made by hybridization of the two
synthetic oligonucleotides, 5`-AATTCTAGACACTGCTCTT-TAACAATTTAG-3` and
5`-GATCCTAAATTGTTAAAGAGCAGTGTCTAG-3` containing the minimal boxA
sequence (Squires et al., 1993) and flanking sequences was
digested with EcoRI and BamHI and inserted into EcoRI-BamHI-digested pUHE23-2. The operation resulted
in the deletion of 33 nucleotide residues from the polylinker region,
containing an unused Shine-Dalgarno sequence, and the insertion of 30
nucleotide residues. The resulting plasmid is called pUV15. pUV15 was
digested with BamHI and PvuII and a 3,100-bp BamHI-DraI fragment containing the entire lacZ reading frame from pTL25 (Linn and Ralling, 1985) was inserted
behind the boxA sequence.
Construction of pUV25
pUV15 was digested with BamHI and PvuII, cutting just behind the boxA
sequence and in the cat gene, respectively. A 3,111-bp BglII-NruI fragment from pA2-1 (Plumbridge and
Springer, 1983) containing the entire infB and a small part of
the 5` part of the 15K gene was inserted behind the boxA
sequence, resulting in an in-frame fusion of 15K` and `cat.Growth Conditions
The bacteria were grown in A
+ B medium (Clark and Maal, 1967) supplemented with
thiamine (1 µg/ml), ampicillin (100 µg/ml), and glucose (0.2%).
CF1648 and CF1693 derivatives were also supplemented with casamino
acids (0.2%), uracil (20 µg/ml), and tetracycline (30 µg/ml).
The bacteria were grown at 37 °C. Isoleucine starvation was induced
by addition of 0.4 mg/ml valine (Leavitt and Umbarger, 1962).
Transcription from P
was induced by 1 mM IPTG.
Induction Lag for
Sampling was performed as described by Vogel and
Jensen (1994b), -Galactosidase
Synthesis
-galactosidase activities were determined as
described by Miller(1972), and the data were analyzed in the square
root plot of Schleif et al. (1973).
RNA Techniques
RNA sampling and purification was
performed as described previously (Vogel et al., 1992); dot
blots and hybridization were performed as described by Vogel and Jensen
(1994a). The dot blots were quantified on an Instant Imager from
Canberra-Packard and subjected to autoradiography. The hybridized
radioactivity (cpm) was depicted as a function of time to give an
induction curve. When the curves were fitted, the earlier time points
were weighted more heavily than the later time points due to
instability of the transcripts (Vogel and Jensen, 1994a). For control
purposes, parallel dot blots were made using a probe complementary to
the bla transcript in each experiments. In vitro transcription of riboprobe plasmids with
[-
P]UTP (DuPont NEN) and plasmids used for
the in vitro transcription have been described previously
(Vogel and Jensen, 1994a; Vogel et al., 1992).
of plasmid pUHE23-2, and it was kept repressed,
until induction, by a lacI
present on an episome.
Transcription was induced at time zero by addition of IPTG, and samples
were withdrawn every 10 s for RNA purification. The RNA samples were
analyzed by dot blot hybridization to the appropriate riboprobes shown
in Fig. 1, and the amount of radioactive riboprobe hybridized to
each RNA sample was quantified on an Instant Imager (Canberra-Packard).
Autoradiograms of two sets of dot blots are shown in Fig. 2.
; T, transcription terminator sequence; `, truncated gene; thicklines, probe complementary to the transcripts
at the indicated positions relative to the 5` end; boxA, the
minimal antiterminator boxA (in the 5` end of the transcripts); AT, the natural antiterminator sequences in the spacer region
of the rDNA in plasmids pUV17 and pUV22.
lacZ mRNA Chain Elongation with and without BoxA
Plasmid
pUV12 contains the normal lacZ gene transcribed from the
P promoter. In pUV16, the minimal boxA sequence was
inserted between the P
promoter and the structural lacZ gene. Fig. 3shows the kinetics of appearance of
full-length lacZ transcripts from these two plasmids following
induction with IPTG. During steady state growth, the transcription time
was 75 s for the normal lacZ gene, corresponding to an
elongation rate of 41 nt/s. When boxA was inserted after the promoter,
transcription elongation occurred considerably faster as the
transcription time was only 46 s, corresponding to 67 nt/s. This
difference in the transcription kinetics for the lacZ gene, in
the presence and absence of boxA, was even more pronounced during the
stringent response (Fig. 3, panelsC and D) since the transcription times for the lacZ mRNA
during the stringent response were 50 s (= 62 nt/s) in the
presence of boxA (pUV16) and 160 s (= 19 nt/s) without the
antiterminator boxA (pUV12). These results indicate that boxA is a
primary determinant of the rate of transcription elongation and of the
lack of sensitivity to ppGpp accumulation during the stringent
response.
Effects of boxA on Transcription Polarity
Fig. 4shows induction experiments with pUV12 and pUV16,
where a probe complementary to the 5` part of the lacZ transcript (see Fig. 1) was used. The slopes of the
induction curves, monitored with this early probe, reflect the relative
initiation rates and differences in the polarity in the very early part
of the gene. There is no difference between the slopes of the induction
curves of the boxA-lacZ transcript (Fig. 4B)
during steady state growth and during the stringent response,
indicating that the initiation rate of the T7 A1 promoter is unchanged
during the stringent response in agreement with earlier observations
that initiation of transcription at the T7 A1 promoter is resistant to
ppGpp in vitro (Kingston et al., 1981). As the
full-length boxA-lacZ transcript also accumulates at similar
rates during exponential growth and amino acid starvation (Fig. 3, panelsB and D), it appears
that amino acid starvation does not induce transcription polarity in lacZ when the boxA sequence is contained in the mRNA. On the
other hand, for the normal lacZ gene on pUV12, it is evident
that amino acid starvation causes a strong transcriptional polarity,
since the slopes of the induction curves probed with the 5`-probe
deviate from each other by 30% (Fig. 4A), while
the slopes of the induction curves probed with the 3`-probe deviate
from each other by a factor of 9 (Fig. 3, panelsA and C).
Effects of BoxA on infB mRNA
In order to ensure
that the effects of boxA on mRNA chain elongation were not specific for
the lacZ gene, we also inserted the antiterminator boxA in
front of infB (plasmid pUV25). The induction kinetics of the
antiterminated infB transcript on plasmid pUV25 are shown in Fig. 5. The results are qualitatively similar to the results
obtained for the antiterminated lacZ gene, since the
transcription elongation rate (70 nt/s) for the antiterminated infB gene during exponential growth was higher than for the infB gene without boxA, i.e. 42 nt/s (Vogel and Jensen, 1994a)
and since it remained high during the stringent response.
Transcription Elongation Rates in a ppGpp
We observed previously that the rate of
transcription elongation increased slightly during the relaxed response
to isoleucine starvation, when the basal ppGpp pool decreased to almost
undetectable levels (Vogel and Jensen, 1994a). This observation
indicated that even basal level ppGpp pools inhibit mRNA chain
elongation. Therefore, we measured the elongation rate on the normal lacZ gene (on plasmid pUV12) and on the antiterminated lacZ gene (on plasmid pUV16) in the ppGppStrain
mutant
CF1693 and the parental strain (Xiao et al., 1991) MG1655
(CF1648). These strains contain a functional chromosomal lac operon, but mRNA synthesis from the chromosomal lacZ gene
was ignored, since dot blots of the later time points after induction
of plasmid free cells only rose by 30 cpm (results not shown), which
constitutes less than 1.5% of the amount of hybridization seen with the
plasmid carrying cells.
relA
spoT derivative (CF1693). The transcription
time of the antiterminated lacZ transcript did not vary
significantly between the two strains as the 5-s difference observed
between the two strains is on the limit of detection in our assay, but
the transcription time of the normal lacZ, gene was 60 s in
the
relA
spoT strain and 80 s in the wild type.
These results confirm that even basal level ppGpp concentrations
inhibit mRNA transcription elongation.
spoT
(A and B) or CF1693
relA
spoT (C and D) harboring either pUV12 (A and C) or pUV16 (B and D). The cells were grown in glucose minimal
medium supplemented with casamino acids and uracil. The experimental
procedures were the same as those for Fig. 3. The experiments
with the
relA
spoT strain were made twice, while the
experiments with the relA
spoT
strain only were performed once in this background
strain.
Effects of a High mRNA Chain Elongation Rate on
Translation
As shown in Fig. 7, the presence of the
antiterminator boxA sequence on lacZ transcript did not affect
the induction lag of -galactosidase, indicating that the rate of
translation is not increased when the rate of transcription is
increased. This result is at variance with the result of Jin et
al.(1992), who observed a 10% reduced induction lag for
-galactosidase in cells harboring the rpoB3595 mutation
that makes RNA polymerase transcribe faster. The difference may be due
to strain differences, but our observation is in accordance with the
finding that RNA polymerase does not seem to limit ribosome movement
during steady state growth (Set al., 1994; Vind et al., 1993).
-galactosidase activity over the uninduced level in MAS90. Filledcircles, pUV12; opencircles, pUV16. MAS90 was grown in glucose minimal
medium. At time zero,
-galactosidase synthesis was induced by
addition of 1 mM IPTG. Samples were pipetted into
chloramphenicol at the indicated times and the activity determined as
described by Miller(1972).
Transcription of rRNA with and without BoxA
The
plasmids described below and in Fig. 1contain different
combinations of a promoter proximal boxA sequence and the boxA sequence
in the spacer region of the ribosomal rrnX operon. In pUV17,
the P promoter is fused to an artificial minimal
antiterminator boxA preceding a segment of rDNA and the 3` part of the cat gene. The rDNA contains the 3` part of the 16 S region,
the spacer region, and the 5` part of the 23 S region, including the
internal antiterminator sequence. In pUV22, the minimal antiterminator
box in front of the rDNA segment was replaced by rDNA, and in pUV26
also the internal antiterminator sequence was removed by deleting 80 bp
from the spacer region.
40 nt/s (see below).
Moreover, as seen from Fig. 8(panels D-F), the
transcription elongation rate from the modified rrnX operon on
any of these plasmids was not reduced during the stringent response as
normally seen for mRNA, indicating either that antiterminator sequences
do not influence ppGpp inhibition or, simply, that the transcribed rDNA
segments in the plasmids do not contain ppGpp specific pause sites.
strain, but higher than the
elongation rate (42 nt/s) of the lacZ mRNA during steady state
growth in the presence of the basal level ppGpp pool.
50% polarity in the infB gene (Vogel and Jensen, 1994a), and no (or extremely little)
polarity in the antiterminator-free rrn-cat transcript (Fig. 8, panelsC and F). We
therefore considered the possibility that the observed changes in the
elongation rate, in response to changes in growth condition, were
secondary results of the changes in transcription polarity (or vice
versa, that the observed levels of polarity were secondary results
of the RNA chain elongation rates). However, we ruled out that
suggestion because the transcription elongation rate on lacZ decreased during the stringent response to isoleucine starvation
and increased during the relaxed response, while the transcriptional
polarity was higher during the relaxed response than during the
stringent response (Vogel and Jensen, 1994a).
-D-galactopyranoside; PCR, polymerase
chain reaction; ppGpp, guanosine 3`,5`-bis(diphosphate).
We thank Michael S(Institute of
Molecular Biology, Copenhagen, Denmark) for strains and plasmids.
Furthermore, we thank Catherine Squires (Tufts Medical School, Boston,
MA) for advice on the antiterminator boxA, V. J. Hernandez (National
Institutes of Health, Bethesda, MD) for the strains CF1648 and CF1693,
and Soumaya Laalami (Pasteur Institute, Paris, France) for the generous
gift of pA2-1.
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, O., eds) pp. 333-337, Academic Press, Inc., New York
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