(Received for publication, August 22, 1996, and in revised form, November 2, 1996)
From the Département de Microbiologie et immunologie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, Québec H3C 3J7, Canada
We have recently found that stable R-loop formation occurs in vivo and in vitro when a portion of the Escherichia coli rrnB operon is transcribed preferentially in its physiological orientation. Our results also suggested that the formation of such structures was more frequent in topA mutants and was sensitive to the template DNA supercoiling level. In the present report we investigated in greater detail the involvement of DNA topoisomerases in this process. By using an in vitro transcription system with phage RNA polymerases, we found that hypernegative supercoiling of plasmid DNAs in the presence of DNA gyrase is totally abolished by RNase H, suggesting that extensive R-looping occurs during transcription in the presence of DNA gyrase. When RNase A is present, significant hypernegative supercoiling occurs only when the 567-base pair rrnB HindIII fragment is transcribed in its physiological orientation. This result suggests that more stable R-loops are being produced in this orientation. Our results also suggest that DNA gyrase can participate in the process of R-loop elongation. The strong transcription-induced relaxing activity of E. coli DNA topoisomerase I is shown to efficiently counteract the effect of DNA gyrase and thus inhibit extensive R-looping. In addition, we found that an R-looped plasmid DNA is a better substrate for relaxation by E. coli DNA topoisomerase I as compared with a non-R-looped substrate.
DNA topoisomerases are enzymes involved in the regulation of DNA
conformation (for a review see Ref. 1). Since the topology of DNA
affects most of its functions, DNA topoisomerases are considered key
elements in the regulation of genomic activities. In bacteria, they are
involved in the control of chromosomal DNA supercoiling (2). This task
is apparently performed by two DNA topoisomerases with opposite
enzymatic activities: DNA topoisomerase I (originally known as protein), encoded by the topA gene that specifically relaxes
negative supercoiling (3), and DNA gyrase encoded by the
gyrA and gyrB genes that introduces negative
supercoiling into the DNA (4). The involvement of DNA topoisomerases in every step of DNA replication, namely initiation, elongation, and
termination is also well known in bacteria (5). Although much is known
about the role of DNA topoisomerases in transcription initiation
through the modulation of DNA supercoiling level (6), little is known
about their involvement in the two following steps, namely elongation
and termination.
The tight association of DNA topoisomerases with actively transcribed genes has been well-described in eucaryotes (7-13). In bacteria, changes in plasmid DNA supercoiling in topoisomerase mutants have been linked to transcription elongation in some cases (14, 15). Hence, the production of positively supercoiled plasmid DNA upon DNA gyrase inactivation (16) is linked to transcription elongation (15). In the absence of the topA gene function, the production of hypernegatively supercoiled plasmid DNA (17) has also been linked to transcription elongation (14). However, only transcription of genes encoding for membrane-bound proteins are shown to generate such hypernegative supercoiling (18). One exception is the galK gene which, upon transcription in cis, can be responsible for hypernegative supercoiling of plasmid DNAs (19). In order to explain the link between DNA topoisomerases and transcription elongation in both eucaryotes and procaryotes, a twin-domain model for transcription has been proposed (20). According to this model, positive and negative supercoiling domains are generated, respectively, ahead and behind the moving RNA polymerase. This model has been supported by much experimental evidence (for a review see Ref. 21). Predicted by this model is the fact that co-transcription and translation with simultaneous attachment of the nascent peptide to the membrane will generate the maximum number of supercoils, because in this situation the rotation of the elongation complex is completely blocked. This model also explains why positive supercoiling will be generated during transcription in the absence of DNA gyrase, since, in this situation, only the negative supercoils will be removed by DNA topoisomerase I. Hypernegative supercoiling in the absence of DNA topoisomerase I is equally explained by the selective removal of positive supercoils by DNA gyrase.
However, recent in vitro experiments have shown that the production of hypernegatively supercoiled plasmid DNA during transcription in the presence of DNA gyrase was linked to R-loop formation (22). In this system, the relaxing activity of DNA topoisomerase I was shown to be strongly activated by transcription and to inhibit hypernegative supercoiling, and hence R-looping. It was thus suggested that the regulation of R-loop formation during transcription was an important role for DNA topoisomerases in Escherichia coli. In this model, DNA topoisomerase I and RNase H, an enzyme specifically degrading the RNA portion of an R-loop, are negative regulators of R-loop formation, while DNA gyrase, with its negative supercoiling activity, favors the formation of such structures. This model is also supported by the fact that the growth defect of topA null mutants is partially complemented by overproducing RNase HI and that a defect in DNA gyrase activity helps the growth of rnhA (encoding for RNase HI) mutants (23). Interestingly, the R-loop structure possesses all the elements to be an excellent substrate for DNA topoisomerase I activity, namely a single-stranded DNA region in proximity of a double-stranded one (24).
In order to better study the putative roles of DNA topoisomerases in the regulation of R-loop formation, it was necessary to isolate DNA sequences that are subject to forming such structures upon transcription. We recently showed that stable R-loop formation occurs during transcription of a portion of the rrnB operon, preferentially in its physiological orientation.1 This study also showed that DNA supercoiling was an important factor for R-loop formation. In the present report, we investigated in greater detail the involvement of DNA topoisomerases and DNA supercoiling in R-loop formation during transcription of this rrnB DNA fragment.
pJP459 and pJP461 have been described.1 Briefly, they are pBluescript II KS derivatives in which the 567-base pair HindIII fragment from the rrnB operon of E. coli was inserted in the reverse (pJP459) or the physiological (pJP461) orientation relative to the vector map numbering. For in vitro transcription experiments, these plasmids were purified by using the Qiagen plasmid kit (Qiagen).
Enzymes and ChemicalsT3 and T7 RNA polymerases were from Stratagene. E. coli DNA gyrase A and B subunits and E. coli DNA topoisomerase I were generously provided by James C. Wang (Harvard University) and Leroy F. Liu (University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School). DNA gyrase was reconstituted by preincubating at 37 °C for 30 min. GyrA (1.42 µg) and GyrB (1.5 µg) subunits together in a total volume of 30 µl of a solution containing 50 mM Tris-HCl (pH 7.5), 0.2 mM EDTA, 5 mM dithiothreitol, 70 mM KCl, and 50% glycerol. 1 µl of this mixture (i.e. about 50 ng each of GyrA and GyrB) was used in a typical in vitro transcription reaction (see below). E. coli RNase HI was kindly provided by R. J. Crouch (National Institutes of Health). RNase A, from Boehringer Mannheim, was prepared as described (25). Proteinase K was from Boehringer Mannheim.
In Vitro Transcription ReactionsTypical in vitro transcription reactions were performed essentially as described previously (22). 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 pJP459 or pJP461 DNA, 10 units of T3 or T7 RNA polymerase, and when specified, RNase A and RNase H were added at the indicated concentrations. Unless otherwise indicated, the reactions were incubated at 37 °C for 3 min before the addition of E. coli DNA gyrase (about 50 ng of each subunit) and then incubated for an additional 10 min at the same temperature. 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 an incubation of 30 min at 37 °C, the samples were extracted once with phenol and once with chloroform and then precipitated with ethanol. They were resuspended in 20 µl of a reaction buffer containing 10 mM Tris (pH 8.0), 10 mM MgCl2, 100 mM NaCl, 1 µg of RNase A, and 20 ng of RNase H. After an incubation of 45 min at 37 °C, the samples were phenol-extracted and then analyzed by one-dimensional agarose gel electrophoresis (0.8%) with chloroquine (15 µM) in 0.5 × TBE (25). After electrophoresis they were either soaked in 1 mM MgCl2 for 1 h before being stained with ethidium bromide or they were dried and prepared for in situ hybridization as described (15). Two-dimensional agarose gel electrophoresis was performed as described previously (22) except that 0.5 × TBE was used instead of TPE during electrophoresis. The chloroquine concentrations used were 15 and 60 µM, respectively, in the first and second dimension. After electrophoresis, gels were dried and prepared for in situ hybridization as described (15).
R-loop formation was recently shown to occur
preferentially when the 567-base pair HindIII fragment from
the rrnB operon of E. coli was transcribed in its
physiological orientation in a topA mutant and in an
in vitro system using phage RNA polymerases.1
This conclusion is based on the fact that RNase H-sensitive gel retardation of plasmid DNAs during electrophoresis was observed when
this fragment was preferentially transcribed in its original orientation, either in vitro or in vivo. R-loop
formation during transcription of this DNA fragment in the
topA mutant was also shown to be sensitive to the DNA
supercoiling level. Previous in vitro studies have shown
that hypernegative supercoiling of pBR322 DNA in the presence of DNA
gyrase and during transcription by E. coli RNA polymerase
was linked to R-loop formation (22). We thus considered the possibility
that the hypernegative supercoiling assay could be used to demonstrate
the orientation preference in the process of R-loop formation during
transcription of the rrnB HindIII fragment (see below). In
addition, in order to study the role of DNA gyrase in R-loop formation
during transcription of the HindIII fragment from the
rrnB operon, it was important to find experimental
conditions allowing both active transcription by phage RNA polymerases
and DNA supercoiling by DNA gyrase. In the beginning we used the pJP459
plasmid, a pBluescript II KS derivative in which the rrnB
HindIII fragment is oriented in such a way that it is transcribed
in its physiological orientation by the T3 RNA polymerase. As can be
seen in Fig. 1, transcription by T3 RNA polymerase
allows the production of hypernegatively supercoiled pJP459 DNA in the
presence of DNA gyrase (complete). The omission of one
nucleotide completely abolished hypernegative supercoiling (3 NTPs),
showing that active transcription is required. Hypernegative
supercoiling of pJP459 DNA seems to be totally dependent on R-loop
formation, since it is completely abolished by the presence of RNase H
during transcription (complete + RNase H). The RNase H
effect is better seen in the bottom panel, where the
hypernegatively supercoiled topoisomers revealed by two-dimensional
agarose gel electrophoresis (complete) are totally absent
when RNase H is added during transcription (RNase H). An
aliquot of the DNA sample loaded on the two-dimensional gel
(complete) was shown to be completely relaxed by E. coli DNA topoisomerase I demonstrating that it is indeed
hypernegatively supercoiled DNA (data not shown). In a separate
experiment, by using relaxed pJP459 DNA, we have shown that the DNA
supercoiling activity of DNA gyrase is not affected at all by the
presence of RNase H under the transcription conditions used (data not
shown). In addition, RNase H had no inhibitory effect on transcription,
and indeed, as previously demonstrated,1 the production of
RNA increased in the presence of RNase H, suggesting that an R-loop can
act as a roadblock for a transcription complex (data not shown).
Interestingly, the presence of RNase A at high concentration had no
effect on the formation of hypernegatively supercoiled pJP459 DNA
(complete + RNase A).
Hypernegative Supercoiling in the Presence of RNase A Occurs Preferentially when the rrnB HindIII Fragment Is Transcribed in Its Physiological Orientation
We considered the possibility that the
hypernegative supercoiling assay might have some advantages over the
gel shift assay to demonstrate R-loop formation. Indeed, in the
hypernegative supercoiling assay, an RNase H-dependent
linking number change that occurs before the arrest of transcription is
taken as evidence for R-loop formation, and it is thus independent of
the stability of the R-loop during electrophoresis. In order to
demonstrate that hypernegative supercoiling occurs preferentially when
the rrnB HindIII fragment is transcribed in its
physiological orientation, time course experiments were performed. In
the beginning, on account of the observation that the production of
hypernegatively supercoiled plasmid DNA was not as fast when T7 RNA
polymerase was used instead of T3 RNA polymerase, we decided to use the
T7 RNA polymerase to facilitate the analysis of the results. pJP459 and
pJP461 DNAs were thus transcribed by T7 RNA polymerase in the presence
of DNA gyrase, and aliquots were periodically taken and mixed with a
stop solution as described under "Experimental Procedures." In
pJP459, T7 RNA polymerase transcribes the rrnB HindIII
fragment in its reverse orientation, whereas in pJP461 this DNA
fragment is transcribed in its physiological orientation by the T7 RNA polymerase. As can be seen in Fig. 2A, the
appearance of hypernegatively supercoiled topoisomers increased with
time and was relatively rapid, since within 2 min a significant amount
of topoisomers were already hypernegatively supercoiled. No differences
were observed between pJP459 and pJP461 DNAs, suggesting that
hypernegative supercoiling, and hence R-looping, was rather nonspecific
under these conditions. However, when RNase A at 20 µg/ml was added during the time course experiment (Fig. 2B), almost no
hypernegatively supercoiled topoisomers were produced when pJP459 was
transcribed (left part), whereas such topoisomers were
clearly produced when the rrnB HindIII fragment was
transcribed in its physiological orientation (pJP461; right
part). These results suggest that the process of R-loop formation
during transcription of the rrnB HindIII fragment in its
physiological orientation is very resistant to RNase A at a high
concentration and therefore that the R-loops are more stable and/or
that they are generated at a higher frequency. We consider unlikely the
possibility that the RNase A effect at this high concentration is
linked to degradation of nascent RNAs that can theoretically
participate in R-loop formation in a 3 to 5
direction, because if
this were the case, then R-loop formation and hence hypernegative
supercoiling should always be sensitive to RNase A at this
concentration, irrespective of the transcribed DNA template. Therefore,
we believe that R-loop formation during transcription by phage RNA
polymerases occurs in a 5
to 3
direction and that the RNA in the
hybrid is elongated by RNA polymerase without being displaced from the
template strand. This is also suggested from experiments described
below.
Highly Negatively Supercoiled Plasmid DNAs Are Produced in the Presence of DNA Gyrase after the Arrest of Transcription
In order
to investigate in greater detail the role of DNA gyrase in R-loop
formation, we designed a two-stage reaction in which DNA gyrase is
added after transcription is arrested. A similar approach has been used
to demonstrate R-loop formation due to transcription of pBR322 DNA in
the presence of DNA gyrase (22). pJP459 and pJP461 DNAs were thus
transcribed with T3 or T7 RNA polymerase, and after the arrest of
transcription by heating, DNA gyrase was added. The results of such
experiments are presented in Fig. 3. Under the
chloroquine concentration used during electrophoresis (7.5 µg/ml;
Fig. 3A), an irregular distribution of topoisomers (e.g. second lane) reflects a population where more
negatively supercoiled topoisomers are present as compared with a
regular distribution of topoisomers (e.g. first lane), where
less negatively supercoiled topoisomers are present. This was confirmed
by two-dimensional agarose gel electrophoresis (Fig. 3, B
and C). As can be seen in Fig. 3A, in the absence
of any added RNases, similar topoisomers distributions are observed,
irrespective of the polymerase and the plasmid DNA used. These
distributions are highly heterogeneous, with some topoisomers being
highly negatively supercoiled. This is better seen in Fig.
3B (pJP459, no RNases) and Fig. 3C
(pJP461, no RNases), where some DNA samples were loaded on a
two-dimensional agarose gel. The fact that RNase H completely
eliminated this heterogeneity and the formation of highly negatively
supercoiled topoisomers shows that this is due to R-loop formation
(Fig. 3, A-C, +RNase H). Interestingly, when
RNase A was added, more negatively supercoiled topoisomers were
produced with T3 RNA polymerase than with T7 RNA polymerase in the case
of pJP459 (Fig. 3, A and B, +RNase A),
whereas the reverse situation was observed for pJP461 (Fig. 3,
A and C, +RNase A). This is expected
if transcription of the rrnB HindIII fragment in its
physiological orientation produces R-loops that are more stable. We
consider it unlikely that this is an indication that R-loop formation
is more frequent in the physiological orientation, since highly
negatively supercoiled topoisomers should be produced in larger amounts
in this orientation, which was clearly not the case (Fig. 3,
B and C, no RNases). The fact that the
formation of highly negatively supercoiled DNAs was completely
abolished by RNase H and only partially by RNase A may suggest that
nascent RNAs are involved in R-loop formation and therefore that DNA
gyrase can participate in R-loop elongation when transcription is
arrested, as suggested previously (22). However, this is not true if,
as stated above, the RNase A effect reflects the stability of the
R-loop and not the presence of free RNAs. Our results also suggest that
longer R-loops are being produced when DNA gyrase is present during
active transcription, since hypernegatively supercoiled plasmid DNAs
are then produced, whereas when DNA gyrase is added after the arrest of
transcription, only highly negatively supercoiled DNAs are produced
(compare the two-dimensional gel of Fig. 1 (left panel) with
the two-dimensional gel of Fig. 3B (no RNases)).
This suggests that extensive R-loop formation occurs only during active
transcription in a 5 to 3
direction and in the presence of DNA
gyrase, as previously suggested in the case of the pBR322 RNAII primer
(26). In this context, the role of DNA gyrase is to counteract
extensive relaxation of plasmid DNAs due to R-loop elongation and
therefore to allow more extensive R-loop elongation. This is because in
the absence of DNA gyrase, extensive R-loop elongation could eventually
be blocked by the accumulation of positive supercoils.
E. coli DNA Topoisomerase I Efficiently Abolishes Hypernegative Supercoiling and Hence R-looping during Transcription in the Presence of DNA Gyrase
Data presented above shows that the hypernegative supercoiling reaction in the presence of DNA gyrase was an efficient way of detecting R-loop formation during transcription. Therefore, this assay can be useful to evaluate the effects of DNA topoisomerase I on R-loop formation during active transcription. Previous in vitro studies have shown that the relaxation activity of E. coli DNA topoisomerase I was strongly activated by transcription (22, 27). This can best be explained by the twin domain model of transcription, where DNA topoisomerase I relaxes negative supercoiling generated behind the moving RNA polymerase. Therefore, it is possible that DNA topoisomerase I directly inhibits R-loop formation by relaxing the local negative supercoiling induced by transcription, if local negative supercoiling is important for R-loop formation. The stimulated relaxation activity of DNA topoisomerase I during transcription can also efficiently counteract the supercoiling activity of DNA gyrase to keep global negative supercoiling of plasmid DNAs below the level required for R-loop formation.
pJP459 DNA was thus transcribed in vitro by T3 RNA
polymerase in the presence of various ratios of E. coli DNA
topoisomerase I to E. coli DNA gyrase. As shown in Fig.
4, 40 ng of DNA topoisomerase I was sufficient to almost
completely inhibit hypernegative supercoiling of pJP459 DNA. 11 ng of
RNase H had the same inhibitory effect (Fig. 4). Therefore, at a
roughly 1:1:1 molar ratio of DNA template, DNA gyrase and DNA
topoisomerase I or RNase H, hypernegative supercoiling was inhibited.
This result suggests that under our experimental conditions, DNA
topoisomerase I was as efficient as RNase H to inhibit hypernegative
supercoiling and hence R-looping in the presence of a similar amount of
DNA gyrase.
The fact that RNase A at a high concentration did not abolish
hypernegative supercoiling (see above), and hence R-looping, suggests
that local DNA supercoiling generated during RNA polymerase movement is
not a key factor for this reaction, at least when a DNA template with a
superhelical density of about 0.05 is used. This is because,
according to the twin domain model, nascent RNA should be required to
create a frictional drag to inhibit RNA polymerase rotation and hence
to allow the generation of negative and positive supercoils during RNA
polymerase movement. In agreement with the model, RNase A at a very low
concentration has been shown to inhibit transcription-induced positive
supercoiling in the presence of E. coli DNA topoisomerase I
(27). Therefore, adding RNase A at high concentrations during
transcription in the presence of both DNA gyrase and topoisomerase I
should favor the hypernegative supercoiling reaction, if the
transcription-generated negative supercoiling is important to stimulate
the relaxation activity of DNA topoisomerase I. This is demonstrated in
Fig. 5, where DNA topoisomerase I and DNA gyrase where
mixed together during transcription of pJP459 DNA by T3 RNA polymerase
in the presence or absence of RNase A at 20 µg/ml. It is evident that
the addition of RNase A favored the hypernegative supercoiling reaction
(compare the left panel (without RNase A) with the
right panel (with RNase A)). According to these results, the
transcription-induced relaxing activity of DNA topoisomerase I
efficiently inhibits hypernegative supercoiling by DNA gyrase by
possibly decreasing plasmid DNA supercoiling below the level required
for R-loop formation.
An R-looped Plasmid DNA Is a Better Substrate for Relaxation by E. coli DNA Topoisomerase I than a Non-R-looped Plasmid DNA
Previous
studies have demonstrated that E. coli DNA topoisomerase I
preferentially binds to the junctions of duplex and single-stranded regions (Ref. 24; for a review see Ref. 1). This explains why this
enzyme will specifically relax negatively supercoiled DNA, since
negative supercoiling favors the unpairing of DNA strands. In this
context, an R-loop could potentially create a hot spot for binding and
relaxation by E. coli DNA topoisomerase I, since such a
structure will create junctions of duplex and single-stranded DNAs.
Therefore, an R-looped plasmid DNA should be a better substrate for DNA
relaxation by this enzyme than a non-R-looped substrate. R-looped
pJP459 DNA was thus prepared by transcription with T3 RNA polymerase.
After the arrest of transcription, only one half of the DNA was treated
with RNase H. It can be seen in Fig. 6 that relaxation
of pJP459 DNA was clearly more efficient when the R-loop was not
eliminated by prior RNase H treatment. Moreover, the topoisomers
distribution was always more heterogeneous, with some topoisomers being
completely relaxed, in the case of the population containing R-looped
plasmid DNAs. The heterogeneity can be explained by the fact that this
population contains both R-looped and non-R-looped plasmid DNAs and
that the R-looped ones are almost completely relaxed before the
non-R-looped substrates are touched by DNA topoisomerase I. According to linking number measurements and gel shift assays,
more than 70% of the plasmid molecules were found to carry R-loops in
this population (data not shown).
In the present report we have investigated the role of DNA topoisomerases in R-loop formation during transcription. Our previous results have indicated that the level of negative supercoiling was an important determinant of R-loop formation in vivo and in vitro.1 This is in agreement with earlier results (28-30). It means that one key role of DNA gyrase is to maintain the negative superhelicity at a level appropriate to allow R-loop formation. In agreement with this result, R-loop formation was not detected in our two-stage reaction when a relaxed DNA template was used during transcription (data not shown). Once R-loop formation is initiated it can elongate if sufficient negative superhelicity is maintained throughout this process. In this context, on a small supercoiling domain such as the pJP459 and pJP461 DNAs used in this study, the role of DNA gyrase is predictable and was indeed observed. Therefore, DNA gyrase will maintain high negative superhelicity throughout the process of R-looping and will produce hypernegatively supercoiled plasmid DNAs upon R-loop removal by RNase H. However, this role of DNA gyrase in R-loop elongation might not be as important in a chromosomal DNA context if only very large supercoiling domains exist (31) and if the diffusion of the supercoils is fast enough. This is because in this context, extensive R-loop elongation will not significantly affect the negative supercoiling level of the domain. However, small supercoiling domains may at least transiently exist, due, for example, to the presence of moving transcription complexes and large DNA-bound protein complexes (20).
We have also investigated the role of E. coli DNA topoisomerase I in R-loop formation. We found that the strong transcription-induced relaxing activity of E. coli DNA topoisomerase I can inhibit R-looping, probably by modulating the global DNA supercoiling level of the DNA template. However, it is still possible that the transcription-induced negative supercoiling might be important for R-loop formation, since half of the global supercoiling is believed to be constrained in vivo by proteins binding to negatively supercoiled DNA (2). In this context, DNA topoisomerase I will control R-loop formation by modulating the transcription-induced negative supercoiling. In addition, we found that the R-loop structure is a hot spot for relaxation by E. coli DNA topoisomerase I. Therefore, relaxation by this enzyme as soon as R-loop formation is initiated may result in the destabilization of the short R-loop. E. coli DNA topoisomerase I can thus indirectly prevent R-loop initiation and directly prevent R-loop elongation, at least in our in vitro system. In this context, it is easy to understand why this enzyme is a key element in the regulation of R-loop formation. Also, since R-loop formation can modulate both DNA gyrase and DNA topoisomerase I activity, it can possibly be an important element in the regulation of the global DNA supercoiling level in vivo. One can also predict that RNase H participates in this function.
We also used the hypernegative supercoiling reaction to investigate the
mechanism of R-loop formation. Together, the fact that hypernegative
supercoiling was only produced during active transcription and was, at
least in some situations, resistant to the presence of RNase A at a
high concentration, suggest that the RNA in the hybrid was never free
and, therefore, that R-loop formation occurred in a 5 to 3
direction.
A similar conclusion was previously reached when RNA primer formation
during pBR322 transcription in the presence of DNA gyrase was studied
(26). However, our results do not exclude the possibility that R-loop formation also occurs from nascent RNA in a 3
to 5
direction during
active transcription in the absence of RNase A. Indeed, previous
studies have suggested that R-loop formation during pBR322 transcription with E. coli RNA polymerase occurred by this
mechanism (22). The fact that hypernegative supercoiling during
transcription in the absence of RNase A was nonspecific but was
specific in the presence of RNase A suggests that some R-loops are more
stable, or persistent, than others. To be biologically meaningful, the stability factor is probably the most important for an R-loop. Therefore, R-loop formation during transcription of the rrnB
HindIII fragment in the physiological orientation is possibly
biologically significant. Indeed, we observed that transcription of
this fragment, preferentially in its physiological orientation, on a
plasmid DNA partially inhibited the growth of a topA mutant
if RNase H was not overproduced.1 Also, our results suggest
that unstable R-loops are frequently produced during transcription and
that DNA gyrase, acting very rapidly, allows their detection.
Considering that the RNase A effect reflects R-loop stability, it may
suggest that our gel retardation experiments, in which the DNA samples
were treated with RNase A at a high concentration prior to
electrophoresis, can also be explained in this context. The fact that
when a hypernegatively supercoiled plasmid DNA was used as a template
during transcription in vitro (data not shown) and in
vivo,1 R-loop formation was still specific to the
physiological orientation of the rrnB fragment, as shown by
the gel retardation assay, can also be explained in the context of
R-loop stability. It may suggest that increasing negative supercoiling
above a certain level does not significantly enhance R-loop
stability.
Although the hypernegative supercoiling reaction can be a good assay to study R-loop formation in vitro, it may not be as useful to study this process in vivo. This is because so many factors can influence DNA supercoiling in vivo and because hypernegative supercoiling can possibly be generated in different ways in vivo (18, 21). One must also consider the possibility that in some situations hypernegative supercoiling occurs before R-loop formation and that the formation of such structures simply protects the hypernegatively supercoiled DNA from relaxation. In vivo, binding of histone-like proteins to DNA, for example, may also protect hypernegatively supercoiled DNA from being relaxed.