From the
The nocP-nocR divergent gene arrangement of
the nopaline catabolism (noc) operon of the Agrobacterium
tumefaciens Ti plasmid pTiT37 was examined with respect to the
expression of the nocP promoter. Under repressive conditions,
i.e. in the absence of nopaline, four distinct levels of
P
Remote operators, which are situated outside of the -65 to
+20 region of the regulated promoter, are found in many
prokaryotic operons, for example in ara, deo,
gal, and lac(1) . These operators, however,
have one or more remote or proximal (between the -65 and +20
positions) counterparts, and interaction of a regulatory protein with
these multiple sites modulates expression of the regulated
promoter
(2, 3, 4, 5) . However, in those
operons where only a single operator is involved, the position of the
operator is proximal to or slightly remote from the promoter, as has
been shown for promoters regulated by the Fur, LexA, MetJ, and PurR
proteins
(1) . In these cases, gene expression is repressed or
activated by direct contact between the regulatory protein and the RNA
polymerase (6). However, regulation of gene expression by a single
remote operator, which is more than 70 bp
In this respect, the position of
a putative operator (O
This report
addresses the role of the putative operator in the regulation of the
nocP promoter. The results demonstrate that in the absence of
the inducer, expression of P
Construction of plasmids pNOC100, pNOC32, pNOC12, and
pNOC12
Induction and measurement of the reporter
enzymes were performed as described previously
(8) , except that
p-nitrophenyl-
To examine their topoisomers,
plasmids pNOC12 and pNOC12
A CATGN
As the NocR protein is not needed for expression of the
isolated nocP promoter, we concluded previously that NocR is a
repressor in the absence of nopaline
(8) . Expression of
P
One possible
explanation for this phenomenon is that competition of closely spaced
promoters, like P
An alternate explanation for the
low level expression of P
To determine how the
abortive transcription from P
The results described earlier in this section suggested that
supercoils generated by the transcription of an adjacent gene may
repress expression of P
We analyzed the expression of the nocP promoter of
the A. tumafaciens Ti plasmid pTiT37, and two main points
should be highlighted from the results described above.
First, we
have found that productive transcription from the adjacent nocR promoter represses expression of the divergent nocP promoter, even in the absence of the NocR regulatory protein. In
previous studies, only activation of a mutant promoter in a
topoisomerase I mutant strain has been reported when a divergent
transcription unit was placed upstream of the
promoter
(26, 27, 28) .
Second, results from
different experiments, in good correlation with each other, revealed
that an upstream sequence, which is the operator for the nocP promoter in the presence of the NocR regulatory protein, modulates
both expression of P
Full repression of the nocP promoter was observed only in the presence of the NocR protein,
its binding site, and productive transcription from the divergent
nocR promoter. Thus it is possible that a mutual interaction
between binding of the NocR protein to the remote operator and DNA
supercoiling has a role in repression of the nocP gene. Our
results suggest that repression of the nocP promoter is
influenced by changes in the local supercoiling level. The sensitivity
of P
In conclusion, our results suggest that gene
expression can be regulated through a single operator at a distance and
that DNA supercoiling is one part of the cellular repertoire by which
gene expression is regulated in naturally found divergent promoter
pairs.
We thank Richard Biggs, Brigitta Dudás, and
Michael McManus for their critical reading of the manuscript and va
Vincze for the plasmid pBI101.3.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
expression were observed. The lowest
level of expression, i.e. full repression, was detected in the
presence of the NocR repressor, together with the remote noc operator and productive transcription from the divergent nocR promoter. The next level was observed in the absence of either the
NocR protein or of the operator or of both. The third level was
detected when abortive transcription from the nocR promoter
occurred, irrespective of the presence or absence of the NocR protein.
The highest level of P
expression was
observed in the absence of both productive transcription from
P
and the operator sequence, whether
or not the NocR protein was present. Under inductive conditions,
i.e. in the presence of nopaline, expression of
P
was activated if both the NocR
protein and the operator were present. Absence of either NocR or the
operator resulted in lack of inducibility of the nocP promoter. Transcription from the divergent nocR promoter
had no influence on the activation of P
. It
was also found that the absence of the operator affected plasmid
supercoiling in vivo. The results suggest that DNA topology
has a role in the regulation of the nocP promoter.
(
)
upstream of the -35 hexamer of a
70 promoter,
has not yet been reported to date.
), which we have
localized in the noc operon of the Agrobacterium
tumefaciens Ti plasmid pTiT37, is unusual. In this operon, two
genes, nocP (formerly called nocB) and nocR,
are divergently transcribed by nonoverlapping promoters. The NocR
protein alternatively represses or activates expression of
P
, depending on the absence or presence of nopaline,
respectively
(7, 8) . The putative operator is located 3
bp downstream of the -10 hexamer of the nocR promoter
and centered 137 bp upstream of the -35 hexamer of the regulated
nocP promoter. Its deletion resulted in failure of NocR
binding in vitro(8) . There are no other binding sites
for the NocR protein in the noc promoter region
(7) ,
and no regulatory sites exist in the coding regions of either the
nocP or the nocR genes
(8) .
is much
higher in the absence than in the presence of the operator, when the
NocR repressor is present. Furthermore, different levels of
P
expression were observed depending
upon productive or abortive transcription from the divergent nocR promoter and presence or absence of the operator sequence, even in
the absence of the NocR regulatory protein. In the presence of the
inducer, P
was activated only in the
presence of both NocR and the operator. Transcription from the adjacent
nocR promoter did not perturb the activation. The noc operator was also found to affect plasmid supercoiling in
vivo. The results suggest that expression of
P
can be controlled through a single
remote operator and that at least repression of the nocP promoter may be interpreted in terms of alteration of local DNA
topology.
o has been described previously
(7, 8) . To
construct pNOC860, the operator-deleted nocR promoter fragment
(P
) was excised from
pNOC12
o by digestion with HindIII+BamHI and
was inserted in front of the promoterless
-glucuronidase
(gusA) gene of pBI101.3
(9) . The
P
-gusA fusion was then cloned upstream of the
P
-luc fusion of pNOC100. The
wild type and the operator-deleted nocR promoters from pNOC12
and pNOC12
o were cloned into pNOC100, yielding pNOC202 and
pNOC301, respectively. For these procedures standard DNA manipulation
techniques were used
(10) . Plasmids were routinely maintained in
Escherichia coli DH5
and were conjugated into the
pTiT37-carrying A208
(11) and the Ti plasmid-free C58C1
(12) A. tumefaciens strains by triparental
mating
(13) .
-D-glucuronide was used as the
substrate for
-glucuronidase.
o were introduced into the E. coli strain HB101. Bacteria were grown in Luria-Bertani media at 37
°C to the mid-log phase and treated with oxolinic acid at 50
µg/ml final concentration for 20 min. Plasmid DNAs were isolated by
the modified alkaline lysis method of Saunders and Burke
(14) .
To separate plasmid topoisomers, electrophoresis of the samples were
done on a 1% agarose gel in TBE buffer
(10) , in the presence of
24 µg/ml chloroquine at 40 V for 20 h at room temperature followed
by staining with ethidium bromide.
CATG direct repeat (Fig. 1) has been
identified as the only binding site for the NocR protein in the
nocP-nocR promoter region and has been proposed to
act as an operator in expression of the nocP promoter
regulated by the NocR protein
(8) . To investigate the role of
this sequence, two plasmids, pNOC32 and pNOC860 (Fig. 2), were
used. Since both plasmids contain the luc and gusA reporter genes fused to the nocP and nocR promoters, respectively, transcriptional activity of the promoters
can be extrapolated by measuring activities of the Luc and GusA
enzymes. Plasmid pNOC32 has the wild type promoter region, whereas in
pNOC860 the putative operator is deleted.
Figure 1:
Sequence
of the nocP-nocR promoter region. The start codons,
the -35 and -10 hexamers are underlined. The
direct repeats of the operator are double underlined. An
alternating purine-pyrimidine sequence overlapping with the operator is
in bold and lowercase labels the base pair which is
out of alternation. The sequence presented here is available from
GeneBank under the accession number
L04475.
Figure 2:
Structure of the pNOC plasmids used in
this study (B). The top part (A) shows the
arrangement of the nocP-nocR region in the Ti plasmid
pTiT37. Genes are labeled by differently shaded boxes. In
pNOC860, pNOC301, and pNOC12o the position of the deleted sequence
is labeled by a black box. The flanking regions of the
plasmids are not shown, and genes are not drawn to
scale.
In the A. tumefaciens strain A208, supplementing the NocR protein from trans, a
15-fold higher expression of P was
detected in the mutant pNOC860 plasmid than in the wild type plasmid
pNOC32, in the absence of nopaline. Since the presence of the direct
repeat is needed for binding of NocR in vitro(8) , the
higher expression of P
of pNOC860 in a
NocR
background could be due to the failure of NocR
binding and indicates the operator role of the CATGN
CATG
sequence.
in the absence of the operator
(strain A208/pNOC860), however, was only 16-17% of the
constitutive expression of the isolated nocP promoter (data
not shown). This observation is contradictory to the rule that deleting
the binding site of a repressor results in constitutive expression of
the regulated gene
(15) . Therefore, we performed the reverse
experiment, i.e. the wild type pNOC32 plasmid was introduced
into a NocR
strain, C58C1. Since the NocR protein
autoregulates its own synthesis
(8) , the 9-fold higher
expression of P
in strain C58C1/pNOC32
() confirmed the absence of the NocR protein. The lack of
the nocR gene in strain C58C1 was also confirmed by Southern
hybridization (data not shown). In this proven NocR
strain, expression of the nocP promoter was similar to
that of strain A208/pNOC860. Furthermore, in the
nocR
O
double mutant C58C1/pNOC860 strain, similar expression of
P
was observed to that which was
detected in the A208/pNOC860 and the C58C1/pNOC32 strains
(). These results revealed that the nocP promoter
is expressed equally in either the absence of the NocR protein or in
the absence of the operator or in the absence of both. Therefore, there
is another factor which caused the relatively low expression of
P
under these circumstances.
and
P
, for the RNA polymerase influences
their expression
(16) . However, despite the 9-fold variation
observed between expression of the nocR promoter in strains
A208/pNOC860, C58C1/pNOC32, and C58C1/pNOC860, only a
1.3-1.5-fold difference was observed between expression of
P
in the same strains. This result
indicates that there is no competition between the nocP and
nocR promoters and that expression of
P
is independent of the strength of
P
.
observed in
the absence of the NocR protein is that the transcription process from
the nocR promoter influences expression of the divergent
nocP promoter. Transcription generates negative supercoils
behind the transcribing RNA polymerase in a topologically closed
domain
(17, 18, 19) , and the generated torsional
stress may affect functions of the nearby
sequences
(19, 20, 21, 22) . The
supercoil-generating effect of the transcription, however, depends on
the transcript length
(23) . To study this aspect in the noc operon, we truncated the gusA gene from the nocR promoter in plasmid pNOC32 to produce plasmid pNOC202
(Fig. 2). As a consequence of the lack of the gusA gene,
transcription from the nocR promoter is abortive in plasmid
pNOC202, resulting in a much shorter transcript compared with plasmid
pNOC32 in which productive transcription occurs. Plasmid pNOC202 was
then introduced into both the NocR
C58C1 and
NocR
A208 strains. A 4.7-fold higher expression of
P
was observed in the absence
(pNOC202) than in the presence (pNOC32) of the gusA gene
(). In the NocR
A208 strain, however,
deletion of the gusA gene resulted in a 47-fold increase of
expression of the nocP promoter (: A208/pNOC202
versus A208/pNOC32). The absolute levels of
P
expression, however, were the same
in either the A208/pNOC202 or the C58C1/pNOC202 strains ().
These results demonstrate that in the absence of the gusA gene, expression of the nocP promoter is independent of
the NocR protein, even in the presence of the operator, and that the
presence of the gusA gene is needed for full repression of
P
by the NocR protein. Furthermore,
the presence of the gusA gene fused to the nocR promoter has reduced expression of the nocP promoter,
even in the absence of the NocR protein.
affects
expression of P
in the absence of the
operator, a plasmid, pNOC301 (Fig. 2), which lacks both the
operator and the gusA gene, was constructed and introduced
into the NocR
A208 and NocR
C58C1
strains. Similar levels of P
expression were detected in either the presence or the absence of
the NocR protein (). The expression of
P
in pNOC301, however, was 3.5-fold
higher than in pNOC202 and about 10-fold higher than in pNOC860, in
either the A208 or the C58C1 strains (). These results
indicated that the enhanced expression of P
in the operator-deleted plasmid pNOC301 is independent of the
NocR protein and simply due to the absence of the operator sequence.
Comparison of the data also revealed that absence of the operator
sequence did not influence expression of P
when the gusA gene is fused to the nocR promoter (pNOC860 versus pNOC32, in strain C58C1). In
contrast, deletion of the operator resulted in a 3.5-fold increase of
P
expression in the absence of the
gusA gene (pNOC31 versus pNOC202, in strain C58C1).
. Furthermore,
the presence of the operator did or did not affect expression of
P
, depending on abortive or productive
transcription of P
, respectively. This
suggested that the operator influences DNA topology, depending on the
supercoil level of the neighboring sequences. To study this, two
plasmids, pNOC12 and pNOC12
o
(8) , containing and lacking
the operator sequence, respectively, were introduced into the E.
coli strain HB101 which is wild type for both gyrase and
topoisomerase I. Plasmid DNAs exist in a negatively supercoiled form
inside the bacterial cells
(24) and can be isolated in this
form. When plasmids pNOC12 and pNOC12
o were isolated from
untreated bacteria, no difference was observed in their topoisomer
distribution (Fig. 3, lanes 1 and 2). In
contrast, treating the strains with a gyrase inhibitor, oxolinic
acid
(25) , resulted in a shift of the topoisomers of both
plasmids (Fig. 3, lanes 3 and 4), indicating
relaxation of the DNA as the consequence of inhibition of gyrase by
oxolinic acid. Despite the relaxation of both plasmids, the
operator-deleted mutant plasmid pNOC12
o became less relaxed
compared with the operator-containing plasmid, pNOC12 (Fig. 3,
lane 3 versus lane 4).
Figure 3:
Effect
of the noc operator on plasmid supercoiling. Lanes 1 and 2, plasmids pNOC12o and pNOC12, respectively,
isolated from untreated E. coli strain HB101. Lanes 3 and 4, plasmids pNOC12
o and pNOC12, respectively,
isolated after oxolinic acid treatment. Direction of the migration is
from top to bottom. At the applied chloroquine
concentration, the topoisomers which were less relaxed (i.e. more supercoiled) before the electrophoresis migrated more
slowly.
Activities of the nocP and
nocR promoters were also determined in all of the constructed
strains under inductive conditions, i.e. in the presence of
nopaline (). The results revealed that the presence of both
the NocR protein and the operator is needed for activation of the
nocP promoter. Absence of either resulted in failure to induce
of P. In contrast to the repression,
activation of P
, however, was nearly
the same in either the presence or the absence of a productive
transcription from the nocR promoter (only a 1.09-fold
difference was observed). Nopaline did not influence expression of the
nocR promoter (), in agreement with our previous
report
(8) .
and DNA
supercoiling, even in the absence of NocR. To our knowledge no similar
effects have been reported for a bacterial operator to date. The
mechanism by which the noc operator affects DNA topology is
not clear yet. However, since an 18-bp alternating purine-pyrimidine
putative Z-DNA forming sequence overlaps the noc operator
(Fig. 1), a B-Z or Z-B transition is possible, as it has been
demonstrated previously that B to Z transition of synthetic
purine-pyrimidine (alternating dG-dC) sequences influences the
supercoil level of DNA
(29) .
to supercoiling is not
unexpected, because it has the features, namely a 19-bp intervening
region between the -35 and -10 hexamers and G-C pairs in
the -10 hexamer (Fig. 1), which have been found in
supercoil-sensitive promoters
(30, 31) . However, in
contrast to the repression, activation of the nocP promoter
was not sensitive to the transcription from the adjacent nocR promoter. This observation suggests that repression and activation
of the nocP promoter may occur by different molecular
mechanisms. This proposal is supported by the observation that
insertion of an extra 47 bp into plasmid pNOC32 between the regulated
nocP promoter and the operator did not influence repression,
but dramatically decreased activation of P
(data not shown).
Table:
Expression of the nocP and nocR promoters in
Agrobacterium
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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Molecular and Cellular Proteomics
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