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INTRODUCTION |
The nascent RNA is normally displaced during the process of
transcription to be translated or to participate in the process of
translation. Shortly after the discovery that cellular DNA can be
negatively supercoiled, it was suggested that the favorable free energy
of such supercoiling should maintain the base pairing between the
nascent RNA and the template DNA strand, and consequently, should
interfere with the process of RNA displacement. Indeed, a direct
correlation between the level of negative supercoiling and the length
of the RNA-DNA hybrid (or R-loop, the template strand is paired with
RNA, leaving the nontemplate strand unpaired) after transcription with
Escherichia coli RNA polymerase was found (1). The formation
of such hybrids was later shown to be due to the denaturing of
transcribing RNA polymerases, and hence to the use of protein
denaturing agents to stop the transcription reactions (2). These
experiments have also demonstrated that the RNA polymerase possesses a
putative "separator" function allowing the nascent RNA to be
displaced as transcription proceeds, and therefore a function that
counteracts the favorable free energy of negative supercoiling for
RNA-DNA hybrid formation. In agreement with this notion are the results
from several experiments revealing that the 9-12-base pair RNA-DNA
hybrid within the RNA polymerase is positioned very close to the
downstream edge of the 18-base pair open transcription bubble (3).
Thus, extensive R-loop formation originating from the short hybrid
within the transcription bubble does not normally occur during transcription.
However, the results of several in vitro and in
vivo experiments have clearly shown that extensive R-loops can
form during transcription on negatively supercoiled templates, and that
both the formation and the length of such structures is modulated by DNA topoisomerases (4-7). These topoisomerases are, DNA gyrase, responsible for the introduction of negative supercoiling, and DNA
topoisomerase I, responsible for the relaxation of negative supercoiling (reviewed in Ref. 8). Indeed, in one study (5), overproduction of RNase H, an enzyme degrading the RNA moiety of an
R-loop, was shown to partially correct the growth defect of
topA null mutants. In another series of experiments, R-loop formation during transcription of a portion of the rrnB
operon, encoding for rRNAs, was shown to occur both in vitro
and in vivo in the absence of DNA topoisomerase I (6, 7).
How these results can be pieced together with the early observations
described above is still unknown. One way to reconcile all these
observations is to consider that the R-loop does not originate from the
transcription bubble but is initiated by the reannealing of a portion
of the nascent RNA with a complementary DNA template region behind the moving RNA polymerase. This, of course, requires that the nascent RNA
be free and therefore not bound by ribosomes and that the corresponding
DNA region behind the moving RNA polymerase be opened. DNA opening
behind the moving RNA polymerase can be nucleotide sequence-dependent and can be promoted by negative
supercoiling generated during transcription in the frame of the
twin-domain model (9). According to this model, supercoiling can be
generated during transcription elongation because of the difficulty for a moving transcription complex to rotate around the double helix. In
this situation, domains of negative and positive supercoiling are
transiently generated, respectively, behind and ahead of the moving
transcription complex. In the absence of DNA topoisomerase I, the local
negative supercoiled domain can build up, whereas the positive one can
be removed by DNA gyrase. Interestingly, we have recently shown that
severe growth inhibition in the absence of DNA topoisomerase I
correlates with transcription-induced supercoiling (10). Because RNase
H overproduction stimulates the growth of the topA null
mutants used to demonstrate this correlation (5), we thought that
R-loop formation generated during transcription might be due to
negative supercoiling generated behind the moving RNA polymerase, which
accumulates in the absence of DNA topoisomerase I. In this report, we
present biochemical and genetic evidence supporting this hypothesis.
Moreover, in agreement with the model for R-loop formation described
above, we present evidence that the binding of ribosomes to nascent
RNAs can inhibit R-loop formation.
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EXPERIMENTAL PROCEDURES |
Plasmids--
The two plasmid DNAs used in this study are pBR322
derivatives and have been described elsewhere (Refs. 10 and 11; also see Fig. 3). Briefly, pBR322
Ptet has a small deletion
within the promoter of the tetA gene that considerably
reduces its expression. In pBR322
tet5' an
HindIII-EcoRV deletion removed the 5' portion of
the tetA gene, so that the remaining tetA RNA is
not translated but still produced. pEM001 and pEM003 are pACYC184
derivatives that, respectively, carry the wild-type rnhA
gene or a mutated version of this gene (7).
In Vitro Transcription Reactions--
Typical in
vitro transcription reactions were performed as described
previously (4, 6). Briefly, they were performed in a volume of 25 µl
of a solution containing 35 mM Tris (pH 8.0), 25 mM MgCl2, 20 mM KCl, 0.4 mM each of CTP, GTP, and UTP, 1.2 mM ATP, 0.5 µg of purified pBR322 DNA, 1 unit of E. coli RNA
polymerase (Amersham Pharmacia Biotech) and, when specified, RNase A
and E. coli RNase HI were added at the indicated
concentrations. The reactions were incubated at 37 °C for 3 min
before the addition (or not) of reconstituted E. coli DNA
gyrase (about 50 ng of each subunit) and then incubated for an
additional 10 min at the same temperature. In Fig. 1, the reactions
were terminated by the addition of EDTA to a final concentration of 30 mM. The samples were brought to 0.3 M NaCl
final before the addition of 100 ng of RNase A, followed by a 60-min
incubation period at 37 °C. The reactions were extracted with phenol
once, chloroform once, and precipitated with ethanol. They were
resuspended in 10 mM Tris (pH 8.0), 10 mM
MgCl2, and 0.1 M NaCl and treated or not with
7.5 ng of RNase H for 60 min at 37 °C. The samples were analyzed by
electrophoresis as indicated. In Fig. 2, the reactions were terminated
by the addition of 25 µl of a solution containing 50 mM
EDTA, 1% SDS, and 12. 5 µg of proteinase K. After 30 min of
incubation at 37 °C, the samples were extracted once with phenol,
once with chloroform, and then precipitated with ethanol. They were
resuspended in 10 mM Tris (pH 8.0), 10 mM
MgCl2, 100 mM NaCl, and treated with 1 µg of
RNase A and 20 ng of RNase H. After 45 min of incubation at 37 °C,
the samples were phenol-extracted and then analyzed by electrophoresis.
Plasmid Extraction for Supercoiling Analysis--
For the
extraction of plasmid DNAs for supercoiling analysis, the following
procedure was used. RFM480 cells
(topA20::Tn10, gyrB221(couR), gyrB203(Ts), Ref. 5)
carrying the various pBR322 derivatives were grown overnight in VB Casa
medium at 37 °C and then diluted 1/75 in prewarmed LB medium. All
the media were supplemented with ampicillin at 50 µg/ml and
chloramphenicol at 30 µg/ml when required. The cells were grown to an
A600 of 0.4 at 37 °C at which time they were
transferred to the desired temperature. The plasmid DNAs were extracted
when the A600 reached about 0.7 (all the strains at 37 °C and only the ones carrying pEM001 at 28 °C and therefore overproducing RNase H) or after an exposition of 2 h at the
respective temperature when an A600 of 0.7 could
not be reached (the strains that do not overproduce RNase H at 28 °C
and all the strains at 21 °C). When hypernegatively supercoiled
plasmid DNAs were produced at 21 °C, the proportion of such
topoisomers reached a maximum after about 1 h at this temperature
and did not change for at least another hour (data not shown). Growth
was stopped by transferring the cells in a tube filled with ice. With
this procedure, the temperature of the cultures immediately dropped to
0 °C. Plasmid DNAs were extracted by an alkaline lysis procedure
(12).
Electrophoresis--
One-dimensional and two-dimensional agarose
gel electrophoresis in the presence or absence of chloroquine were
performed in 0.5 × TBE as described (7). After electrophoresis,
the gels were either stained with ethidium bromide and photographed
under UV light (Figs. 1 and 2) or dried and prepared for in
situ hybridization (Figs. 4-6) as described (7).
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RESULTS |
Extensive R-loop Formation during in Vitro Transcription with E. coli RNA Polymerase Occurs Only in the Presence of DNA Gyrase--
To
study how DNA supercoiling affects R-loop formation during
transcription with E. coli RNA polymerase, we performed
in vitro transcription experiments with a supercoiled DNA
template in the presence or absence of DNA gyrase. The DNA template
used, pBR322, was extracted from a wild-type strain and therefore had a
higher level of negative supercoiling than DNA from topA
null mutants with gyr mutations. Moreover, its effective
negative supercoiling density was significantly higher than DNA from
wild-type cells, considering that about half of the DNA supercoiling is
constrained in vivo (8). To detect R-loop formation, we used
the previously described assay in which RNase H-sensitive gel
retardation and/or relaxation of plasmid DNAs after transcription is
revealed following electrophoresis and ethidium bromide staining of the
gel (7). In addition, all the in vitro reactions were
arrested with EDTA and treated with RNase A before being
phenol-extracted. This procedure was used, because R-loop formation
involving nascent RNA (sensitive to RNase A) can be induced following
denaturing of E. coli RNA polymerases that transcribe DNA
templates with a wild-type supercoiling level (Ref. 2 and data not
shown). The results shown in Fig. 1
clearly demonstrate that significant and stable R-loop formation exclusively occurs when DNA gyrase is present during the transcription reaction. Indeed, RNase H-sensitive gel retardation of plasmid DNAs is
only detected when DNA gyrase was added during transcription (compare
lane 4,
RNase H with lane 5, +RNase H after
transcription, lane 7, +RNase H during transcription or
lane 8, +RNase H during and after transcription). This
alteration in the electrophoretic mobility is better seen in the gel
containing chloroquine (Fig. 1B, lane 4), because
plasmid DNAs carrying R-loops are hypernegatively supercoiled (see
below). Hypernegatively supercoiled plasmid DNA represents a population
of topoisomers that can no longer be resolved by electrophoresis in
agarose gels containing chloroquine (11, 13). These in vitro
results demonstrate that an effective global negative supercoiling
level, even higher than the effective level existing in topA
null mutants, is unable on its own to trigger the formation of stable
R-loops. This is in agreement with the results of early experiments
(2). Most likely, R-loop initiation during transcription by E. coli RNA polymerase involves transcription-induced supercoiling,
and DNA gyrase participates in the process of R-loop elongation to
generate stable and detectable R-loops.

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Fig. 1.
R-loop formation during in vitro
transcription with E. coli RNA polymerase.
The transcription reactions were performed as described under
"Experimental Procedures." When indicated, 7.5 ng of RNase H were
added during transcription. Samples in lanes 5 and
8 were treated with RNase H after transcription. Samples
were analyzed by electrophoresis in agarose gels in the presence
(panel B) or absence (panel A) of chloroquine at
1 µg/ml.
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We next wanted to study more precisely the link between hypernegative
supercoiling and R-loop formation during in vitro
transcription with E. coli RNA polymerase, in the presence
of DNA gyrase. Fig. 2 shows the results
of an experiment in which the susceptibility of hypernegative
supercoiling formation to RNase A and/or RNase H treatments was
evaluated. It can be seen that both RNase A and H dramatically reduced
the production of hypernegatively supercoiled pBR322 DNA in the
presence of DNA gyrase (compare lane 1, complete with
lane 2, RNase H and lane 3, RNase A).
Interestingly, RNase A and H together completely abolished
hypernegative supercoiling (lane 6). This result may suggest
that two independent mechanisms are operating to generate hypernegative
supercoiling; one directly linked to the twin-domain model (sensitivity
to RNase A) and the other one linked to R-loop formation independent of
the twin-domain model (sensitivity to RNase H). However, by a more
careful look at lanes 2 and 3, it is clearly
revealed that both independent treatments to RNase A and H abolished
more than half of the amount of hypernegatively supercoiled DNA
generated in the absence of RNases (lane 1). Moreover, by
increasing the amount of RNase H we found that it is possible to
completely abolish hypernegative supercoiling (data not shown).
Therefore, hypernegative supercoiling during transcription by E. coli RNA polymerase in the presence of DNA gyrase is completely
dependent on R-loop formation, involving the participation of nascent
RNA. In this context, the nascent RNA may serve two purposes: it is
involved in generating negative supercoiling during transcription
according to the twin-domain model, as previously shown in similar
in vitro systems (4, 14), and it anneals with the
complementary DNA template strand to form the R-loop. This is in
contrast with what has been demonstrated for hypernegative supercoiling
during transcription with phage T3 and T7 RNA polymerases (6). Indeed,
in these cases, the formation of such topoisomers was more sensitive to
RNase H treatment but was highly resistant to RNase A treatment,
suggesting that R-loop formation did not involve free RNA and occurred
in the 5' to 3' direction, and therefore the newly synthesized RNA was never displaced from the template strand (6). It is worth mentioning that the natural DNA template for T3 and T7 RNA polymerases is not
supercoiled but linear. Perhaps these polymerases do not possess an
efficient RNA-DNA hybrid separator function as found for E. coli RNA polymerase, to counteract the favorable free energy for R-loop formation during transcription of a negatively supercoiled template. Indeed, stable R-loop formation on supercoiled templates during transcription by T3 and T7 RNA polymerases is detected in the
absence of DNA gyrase, as opposed to the situation with E. coli RNA polymerase (Fig. 1, lane 3), and under the
same experimental set-up as the one used in this study
(7).1

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Fig. 2.
Hypernegative supercoiling during in
vitro transcription with E. coli RNA
polymerase. The transcription reactions were performed as
described under "Experimental Procedures." When indicated, 7.5 ng
of RNase H and/or 1 µg of RNase A were added during transcription. 3 NTPs means that GTP was omitted during transcription. pBR322 represents
a sample of the plasmid DNA used in this study. The samples were
analyzed by electrophoresis in an agarose gel containing 7.5 µg/ml of
chloroquine.
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R-loop-dependent Hypernegative Supercoiling of Plasmid
DNAs in topA Null Mutants Occurs When DNA Gyrase Is Very Active during
Transcription and in the Absence of Translation--
Our next goal was
to reproduce the in vitro data presented above in
vivo, and to test the model for R-loop formation involving free
nascent RNA and transcription-induced supercoiling. Because R-loop
formation can be induced on a negatively supercoiled template by the
use of protein denaturing agents during nucleic acids extraction, we
decided to use the RNase H-sensitive hypernegative supercoiling assay
to reveal R-loop formation in vivo. The generation of such topoisomers occurs within the cells, and it is therefore a more reliable assay to reveal R-loop formation in vivo. Because
all the previous in vitro and in vivo studies
have shown that detectable R-loop formation does not occur in the
presence of DNA topoisomerase I, our in vivo experiments
were performed in a topA null mutant. In addition, because
the presence of cellular RNase H could potentially be a problem, we
used a topA null mutant that grows better when RNase H is
overproduced (5). This topA null mutant, RFM480, carries the
topA20::Tn10 allele and a
gyrB(Ts) allele allowing the modulation of cell growth in a
manner that depends on the temperature. Low temperatures (30 °C and
below) are more restrictive for this strain, owing to this
gyrB(Ts) allele that regains a more wild-type level of
activity under these conditions. This explains why the growth of RFM480
is cold-sensitive. At these temperatures, the topA null
mutant behaves as a true topA mutant without compensatory
mutations, and its growth is shown to be stimulated by overproducing
RNase H (5).
The plasmid DNAs used in our studies are pBR322 derivatives. One
derivative, pBR322
Ptet (Fig.
3), has a small deletion within the
tetA promoter region that was originally believed to abolish tetA gene expression (11). This plasmid was used to show
that the formation of hypernegatively supercoiled pBR322 was linked to
tetA gene expression, because such topoisomers were not
detected when it was extracted from a widely used topA null
mutant, DM800 (11). The formation of hypernegatively supercoiled pBR322
DNA was later shown to be due to membrane anchorage of the
transcription complex via the tetA gene product, a membrane
bound protein (15). We have recently found that
pBR322
Ptet confers low level tetracycline resistance,
because a weak promoter was reconstituted during the construction of
this plasmid. When this plasmid was extracted from our cold-sensitive
topA null mutants exposed to nonpermissive temperatures,
transcription-dependent hypernegatively supercoiled plasmid
DNA was detected (10). This result provided evidence that severe growth
inhibition of topA null mutants correlates with
transcription-induced supercoiling but not with global supercoiling. We
found that an EcoRI-EcoRV deletion within pBR322
that totally abolishes tetracycline resistance and tetA gene
expression, also almost completely abolished the formation of such
topoisomers (data not shown). This result suggests that the generation
of hypernegatively supercoiled pBR322
Ptet DNA is due to
the residual tetA gene expression originating from that
plasmid. We found that RNase H overproduction, conferred by the
presence of the multicopy plasmid pEM001 that carries the
rnhA gene, had no effect on the formation of such
topoisomers (data not shown). This means that R-loop formation is not
involved in hypernegative supercoiling of pBR322
Ptet DNA.
According to our model for R-loop formation, this is not a surprising
result, because the tetA mRNA from that plasmid is
translated, and therefore the nascent RNA is not free to hybridize with
the template DNA strand. Results presented below support this
conclusion.

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Fig. 3.
The 5' tetA gene region of
the various pBR322 derivatives used in this study. The 5'
tetA region of A (pBR322), B
(pBR322 Ptet) and C
(pBR322 tet5'). 35 and 10
indicate the nucleotide sequence determinants for 70 E. coli promoters; +1 refers to the transcription
initiation site; RBS to the ribosome binding site
(Shine-Dalgarno sequence); and ATG to the initiation codon
for the TetA protein. The promoter activity of the tetA gene
of pBR322 tet5' was confirmed when the lacZ
gene was cloned downstream in the appropriate orientation.
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The other pBR322 derivative used in the present study is
pBR322
tet5', from which the remaining portion of the
tetA gene is transcribed but the resulting RNA is not
translated (Fig. 3). This is because the
HindIII-EcoRV deletion that was made to construct this plasmid eliminated the 5' part of the tetA gene
including the original
10 promoter region, the Shine-Dalgarno
sequence (ribosome binding site), the ATG initiator codon, and one
transmembrane domain responsible for the anchorage of the TetA protein
to the membrane. However, an active promoter was reconstituted (Fig. 3). This promoter is almost as active as the original tetA
promoter, according to lacZ assays (data not shown). When
this plasmid DNA was extracted from our cold-sensitive topA
null mutants exposed to nonpermissive temperatures,
transcription-dependent hypernegatively supercoiled
topoisomers were detected (10). The formation of such topoisomers is
linked to tetA gene transcription, because, as mentioned
above, a larger deletion, EcoRI-EcoRV (Fig. 3),
which completely eliminates tetA gene transcription, also
dramatically reduces the accumulation of such topoisomers. The next
series of experiments was performed to verify if the production of
hypernegatively supercoiled pBR322
tet5' DNA was linked to
R-loop formation. For that purpose, we introduced an additional plasmid
DNA, pEM001, carrying the rnhA gene or the control plasmid,
pEM003, carrying an inactivated rnhA gene, within RFM480
bearing pBR322
tet5'. The cells were grown in LB medium at
37 °C and exposed to the indicated temperatures as described under
"Experimental Procedures." The extracted plasmid DNAs were
subjected to electrophoresis in agarose gel in the presence of
chloroquine at 7.5 µg/ml. Under these conditions the more negatively
supercoiled topoisomers migrate slowly except for the fastest migrating
band pointed out by an arrow (Fig. 4, [-
-]), which represents hypernegatively supercoiled plasmid DNAs. First
of all, it can be seen that the global DNA supercoiling level in the
various strains, represented by the topoisomers distributions of
pBR322
Ptet DNA without considering hypernegatively
supercoiled DNA, increases as the temperature decreases. This is
expected, because the temperature-sensitive DNA gyrase becomes more
active at low temperatures. It is also obvious that RNase H
overproduction did not have any effect on global supercoiling level
(for example, compare lane 5, +pEM001 with lane
6, +pEM003). Results presented in Fig. 4 also clearly demonstrate
that, as opposed to pBR322
Ptet DNA, RNase H
overproduction abolished the generation of hypernegatively supercoiled
pBR322
tet5' DNA (compare lane 5 with
lane 6, 28 °C and lane 8 with lane
9, 21 °C). Most likely, such topoisomers are not produced at
37 °C, because DNA gyrase activity for R-loop elongation is too weak to counteract the wild-type level of RNase H activity. As the temperature decreases, DNA gyrase regains a higher level of activity, and the wild-type level of RNase H activity is no longer sufficient to
completely abolish extensive R-loop formation; hence, the generation of
hypernegatively supercoiled pBR322
tet5' DNA.
Two-dimensional agarose gel analysis was also performed to confirm the
presence of hypernegatively supercoiled pBR322
tet5' DNA
and the fact that its formation is abolished by overproducing RNase H
(Fig. 5, top panels). Fig. 5
also shows that RNase H overproduction partially abolished the
formation of hypernegatively supercoiled pACYC184
tet5' (bottom panels), a pACYC184 derivative carrying an identical
deletion to the one found in pBR322
tet5'. The
tetA gene is the only common DNA sequence between pACYC184
and pBR322. As it is the case for pBR322 when the tetA gene
of pACYC184 is translated, the formation of hypernegatively supercoiled
plasmid DNA is not sensitive to RNase H overproduction (data not
shown).

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Fig. 4.
The formation of RNase H-sensitive
hypernegatively supercoiled plasmid DNAs in a cold-sensitive
topA null mutant, one-dimensional gel analysis.
RFM480 cells carrying pBR322 tet5' were grown, and the
plasmid DNAs were extracted as described under "Experimental
Procedures." In lanes 1, 4, and 7,
the cells carry no additional plasmid DNA, whereas in lanes
2, 5, and 8 they also carry pEM001, and in
lanes 3, 6, and 9, they also carry
pEM003. + indicates that RNase H was overproduced (the cells carrying
pEM001). [- -] indicates hypernegatively supercoiled plasmid DNAs.
The samples were analyzed by electrophoresis in an agarose gel
containing 7.5 µg/ml of chloroquine. The gel was probed with a
32P-labeled DNA fragment carrying the bla gene
of pBR322.
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Fig. 5.
The formation of RNase H-sensitive
hypernegatively supercoiled plasmid DNAs in a cold-sensitive
topA null mutant, two-dimensional gel analysis.
RFM480 cells carrying either pBR322 tet5' (top
panels) or pACYC184 tet5' (bottom panels)
were grown, and the plasmid DNAs were extracted as described under
"Experimental Procedures." The cells also carry either pEM001
(top left panel), pEM003 (top right panel),
pSK760 (bottom left panel), or pSK762c (bottom right
panel). pSK760 and pSK762c are, respectively, the equivalents of
pEM001 and pEM003 but contain a ColE1 origin of replication that is
compatible with pACYC184 tet5' (5). The samples were
analyzed by two-dimensional agarose gel electrophoresis. The
chloroquine concentrations used were 7.5 µg/ml and 30 µg/ml,
respectively, in the first and second dimension. Under the chloroquine
concentrations used, hypernegatively supercoiled plasmid DNAs migrate
at the end of the left part of the curve. When
pBR322 tet5' was analyzed, the gel was probed with a
32P-labeled DNA fragment carrying the bla gene
of pBR322. When pACYC184 tet5' was analyzed, the gel was
probed with a 32P-labeled DNA fragment carrying the
cat gene of pACYC184.
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One prediction that could be made regarding hypernegatively supercoiled
pBR322 DNA, is that the formation of such topoisomers should be
insensitive to protein synthesis inhibitors under conditions where DNA
gyrase is active enough during transcription to promote R-loop
formation. This is demonstrated by the experiment shown in Fig.
6. Indeed, the protein synthesis
inhibitor spectinomycin abolished the formation of hypernegatively
supercoiled pBR322 only under conditions where DNA gyrase was not very
active, therefore when RFM480 cells were grown at 37 °C (Fig. 6,
compare lane 1 with lane 3, respectively, + or
spectinomycin). Under such conditions, hypernegative
supercoiling has been shown to be dependent on membrane anchorage of
the transcription complex via the TetA protein (15). However, when
RFM480 cells were exposed to 21 °C, hypernegative supercoiling was
not abolished by spectinomycin treatment as expected, because at this
temperature DNA gyrase is active enough to promote R-loop-dependent hypernegative supercoiling (Fig. 6,
compare lane 2 with lane 4, respectively, + or
spectinomycin). When a similar experiment was performed with
the pBR322 derivative carrying the EcoRI-EcoRV
deletion that completely inactivates tetA gene expression, only a very small amount of hypernegatively supercoiled plasmid DNAs
could be detected (data not shown). This result suggests that
R-loop-dependent hypernegative supercoiling of pBR322 in the absence of translation is mostly related to tetA gene
transcription.

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Fig. 6.
Sensitivity of hypernegative supercoiling of
pBR322 DNA to the protein synthesis inhibitor, spectinomycin.
RFM480 cells carrying pBR322 were grown to an
A600 of 0.4 at which time spectinomycin (500 µg/ml) was added (lanes 1 and 2) or not
(lanes 3 and 4). The cells were incubated for an
additional 15 min at 37 °C before being exposed to 21 °C. An
aliquot of cells was rapidly withdrawn for plasmid DNAs extraction
(lanes 1 and 3). The cells were incubated for an
additional 2 h before the second plasmid DNAs extraction
(lanes 2 and 4). Note that the topoisomers
distribution is more bimodal (hypernegative supercoiling and global
supercoiling) when translation is inhibited (compare lane 2 with lane 4). This is observed when hypernegative
supercoiling is R-loop-dependent (Fig. 4). When translation
is not inhibited, the topoisomers distribution is more heterogeneous
and continuous, as previously shown for pBR322 DNA extracted from
various topA mutants (11, 13, 15).
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All together, the results of our in vivo experiments are in
accordance with the model for R-loop formation during transcription by
E. coli RNA polymerase. 1) RNase H-sensitive hypernegatively supercoiled plasmid DNA, and hence R-loop formation, is detected under
conditions where DNA gyrase is very active during transcription. Indeed, under these conditions, very weak tetA gene
expression from pBR322
Ptet DNA is still sufficient to
trigger the formation of hypernegatively supercoiled plasmid DNA. 2) It
is only in the absence of translation that RNase H-sensitive
hypernegatively supercoiled plasmid DNA, and hence R-loop formation, is
detected. This is also in agreement with our previous results showing
RNase H-sensitive hypernegative supercoiling when a portion of the
untranslated rrnB operon was transcribed (7).
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DISCUSSION |
Two major conclusions emerge from the work presented here. First,
R-loop formation is linked to transcription-induced supercoiling but
not to global supercoiling level. This is in agreement with the results
demonstrating that extensive R-loop formation does not normally occur
during transcription on negatively supercoiled templates unless
E. coli RNA polymerase is denatured (Ref. 2 and data not
shown). This result shows that even under conditions where the negative
supercoiling level favors R-loop formation, they do not form. It may
suggest that DNA supercoiling is not a major contributor to R-loop
formation when such structures are generated. The best studied example
of R-loop formation involves the origin of replication of the ColE1
plasmid DNA. In these studies, R-loop formation was clearly shown to be
nucleotide sequence-dependent (16, 17). However, the
results of several in vitro and in vivo
experiments, including the work presented here, have clearly shown that
extensive R-loops can form during transcription and that it is
regulated by DNA topoisomerases that modulate the supercoiling level in
E. coli (4-7). Because the global supercoiling level does
not seem to be involved in R-loop formation, one obvious alternative
explanation for the contribution of DNA topoisomerases in this process
is in relation to transcription-induced supercoiling in the frame of
the twin-domain model (9). Our results support this interpretation. The
model derived from this interpretation implies that the free nascent
RNA, in addition to being directly involved in RNA-DNA hybrid
formation, also contributes to the generation of negative supercoiling
during transcription. Such supercoiling can promote DNA opening behind
the moving RNA polymerase, which is a prerequisite to the initiation of
the annealing between the nascent RNA and the corresponding DNA
template region. DNA opening could also be promoted by specific DNA
sequences and/or global supercoiling level. Once the R-loop is
initiated, it creates an anchor for the moving RNA polymerase,
inhibiting its rotation and hence increasing transcription-induced
negative supercoiling. This supercoiling can promote R-loop elongation
if it is not relaxed by DNA topoisomerase I. Indeed, anchorage of the
RNA polymerase via the annealing of the nascent RNA with the DNA
template strand was originally described as one potential mechanism for
increasing transcription-induced supercoiling (9). Anchoring the RNA
polymerase will also increase transcription-induced positive
supercoiling. Such supercoiling must be removed in order for the RNA
polymerase and R-loop extension to progress at a proper rate. DNA
gyrase will be involved in relaxing positive supercoiling, and
therefore will contribute to R-loop elongation. DNA gyrase can also
promote R-loop elongation by constantly replacing the negative
supercoils removed by this process. Either way, DNA gyrase must be
active enough to counteract the cellular RNase H activity that disrupts the R-loop. Therefore, if DNA gyrase is active enough, extensive, stable, and detectable R-loops will be generated. In the absence of
RNase H, a mutated DNA gyrase, which even causes a decrease in global
negative supercoiling below the wild-type level, will be sufficient for
R-loop elongation. This is supported by the fact that double
topA-rnhA mutants are nonviable (5) even when the
strain carries very good compensatory gyr mutations, as in the case for the widely used DM800 topA null
strain.1 Moreover, the growth of RFM480 in rich media is
stimulated by RNase H overproduction even when global negative
supercoiling is below the wild-type level (growth at 37 °C) (5).
Interestingly, in vitro transcription experiments with
E. coli RNA polymerase from synthetic RNA-DNA bubble
duplexes have shown that the nascent RNA was frequently rehybridizing
to the permanently unpaired DNA bubble (18). In the frame of our model
for R-loop formation, the presence of this permanent bubble can be
viewed as an optimal condition for R-loop initiation.
An important question that was not addressed in this study is related
to RNA swiveling, which is required for extensive R-loop formation. In
fact, the length of the R-loop when the RNA polymerase is still present
on the template may not be limited by energetic considerations related
to negative supercoiling, but rather by the capacity of the RNA to
swivel. It is possible that the initiated R-loop behind the moving RNA
polymerase eventually extends up to the transcription bubble. Under
these conditions, the separator function of the RNA polymerase might be
disrupted. In this context, swiveling of the RNA-DNA hybrid within the
transcription bubble should be sufficient to allow the progression of
transcription and R-loop elongation. This model can also explain the
synthesis of the RNA primer at the ColE1 origin of replication (17).
However, additional experiments are required to solve this problem.
The second important conclusion emerging from this study concerns the
role of DNA topoisomerase I in E. coli. In a previous study
(10), it was shown that severe growth inhibition of topA null mutants correlates with transcription-induced supercoiling. Together with the results presented in this study and the ones showing
that RNase H overproduction stimulates the growth of topA null mutants (5), we can conclude that one major function of DNA
topoisomerase I is to relax transcription-induced negative supercoiling
to inhibit R-loop formation.