From the Department of Biophysics and Biochemistry,
Graduate School of Science, University of Tokyo, Hongo,
Tokyo 113, Japan and the
Department of Applied Physics and
Chemistry, University of Electro-Communications, Chofu-shi,
Tokyo 182, Japan
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ABSTRACT |
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The autonomous replication region of
plasmid ColIb-P9 contains repZ encoding the RepZ
replication protein, and inc and repY as the
negative and positive regulators of repZ translation,
respectively. inc encodes the antisense Inc RNA, and
repY is a short open reading frame upstream of
repZ. Translation of repY enables
repZ translation by inducing formation of a pseudoknot
containing stem-loop I, which base pairs with the sequence preceding
the repZ start codon. Inc RNA inhibits both
repY translation and formation of the pseudoknot by binding
to the loop I. To investigate control of repY expression by
Inc RNA, we isolated a number of mutations that express
repY in the presence of Inc RNA. One class of mutations
delete a part of another stem-loop (II), which derepresses
repY expression by initiating translation at codon 10 (GUG), located within this structure. Point mutations in stem-loop II
can also derepress repY translation, and the introduction
of compensatory base-changes restores control of repY
translation. These results not only indicate that suppressing a cryptic
start codon by secondary structure is important for maintaining the
translational control of repZ but also demonstrate that the
position of start site for repY translation is critical for
its control by Inc RNA. Thus, Inc RNA controls repY
translation by binding in the vicinity of the start codon, in contrast
to the control of repZ expression at the level of loop-loop interaction.
The initiation phase of translation is rate-limiting in protein
synthesis and often the target of control of gene expression (1). It is
well established that the selection of translational start sites in
prokaryotes is stimulated by base pairing between the 3'-end of the
small ribosomal RNA (16 S rRNA) and the Shine Dalgarno
(SD)1 sequence in the
mRNA, located ~7 bases upstream of the initiation codon (2). The
ribosome-binding site (RBS) of the mRNA contains the SD sequence
and the initiation codon (3). Translational control can be achieved by
sequestering or opening the RBS, either by binding of
trans-acting factors (RNA or protein) or by modulating the
higher-ordered structure of the mRNA (2). We have been studying the
various aspects of translational control found in the replication
control system in plasmid ColIb-P9 (4).
The replication of the ColIb-P9 plasmid (93 kilobases, IncI Inc RNA represses the translation of both repY and
repZ at different rates (9). In the presence of Inc RNA, the
level of repZ expression is kept constant regardless of
changes in copy number of the repZ reporter plasmid,
although repY expression is increased with an increase in
plasmid copy number (10). Thus, Inc RNA appears to help maintain the
constant level of repZ expression, by repressing
repZ expression more efficiently than repY
expression. Because the level of repZ expression is linearly
correlated with the copy number of ColIb-P9, it was proposed that the
negative feedback loop conferred by this differential control plays the major role in establishing the constant copy number of ColIb-P9 (4).
To understand the mechanism of this unique differential control by Inc
RNA, it was important to analyze the structure of the repZ
mRNA leader region and its changes during the regulation. Fig.
1B shows the secondary structures of the repZ
mRNA leader, deduced from the ribonuclease cleavage experiments
in vitro (10). We found that two stem-loop structures, I and
III, play important negative roles. Structure I is the target site of
the Inc RNA: base pairing between the 5'-rGGC-3' sequence in the loop I
(positions 327-329) and its complementary sequence in the Inc RNA is
rate-limiting for the binding of Inc RNA (11). Structure III sequesters
the repZ RBS (Fig. 1B): its disruption by
mutations derepresses repZ expression, independently of the
actions of repY and Inc RNA (8). These stem-loop structures
are drawn schematically in Fig. 1C, panel a.
The third structural element is the psuedoknot formed with
structure I and the base pairing between the 5'-rGGCG-3' (loop I at
positions 327-329) and 5'-rCGCC-3' sequences (stem III at positions
437-440) (bracketed in Fig. 1B). The formation
of this pseudoknot is absolutely required for repZ
translation (4, 9, 10) and is induced in vitro when
structure III is disrupted by mutations (10). We proposed that the
translation of repY induces the pseudoknot formation during
the termination process, thereby stimulating the access of the ribosome
to the repZ RBS (Fig. 1C, panel b), whereas Inc
RNA inhibits it immediately by binding to the loop of structure I (Fig.
1C, panel c). Subsequently, duplex formation between the
5'-end of Inc RNA and a region proximal to the repY RBS
inhibits repY translation (Fig. 1C, panel d). We
provided the biochemical evidence for the loop-loop inhibition model in
panel c by showing that the pseudoknot formation and the
initial base paring of Inc RNA to the loop I are competitive (4).
Importantly, the model in Fig. 1C can explain the
differential control of repY and repZ expression
by Inc RNA.
IncFII plasmids also encode both antisense RNA and leader peptide
upstream of their replication initiator genes as negative and positive
regulatory elements, respectively (Refs. 12 and 13; for review, see
Ref. 14). In the case of plasmid R1 of this group, it was demonstrated
that the bound antisense CopA RNA inhibited the access of 30 S ribosome
to the leader peptide RBS in vitro (15). However, these
plasmids do not require mRNA pseudoknot for the expression of their
replication initiator genes (13, 16). Thus, the IncI Here, we study the regulation of repY expression by Inc RNA
through isolating a number of mutations that derepress repY
expression in the presence of Inc RNA. The members of one class of
mutations delete a part of the stem-loop structure II (see Fig.
1B) and allow repY translation from its codon 10, GUG, normally embedded in this structure. Point mutations in structure
II can also derepress repY translation, and the introduction
of compensatory base-changes restores control of repY
translation. These results indicate that the position of the start site
for repY translation is critical for control by Inc RNA. In
contrast, the control of repZ expression by Inc RNA is not
constrained spatially, because the site of Inc RNA binding is ~90
bases upstream of the RepZ start codon. Furthermore, by identifying the
importance of structure II for suppressing a cryptic start codon, we
strengthen the idea that mRNA structure is critical for maintaining
the integrity of repZ translational control.
Bacteria, Phages, and Plasmids--
The Escherichia
coli K-12 strains W3110 and W3110(
Plasmids used in this study are listed in Table
II. pCH11 and its derivatives were used
as the donor of Inc RNA in vivo, and as the template for
in vitro synthesis of Inc RNA and RepZ protein. pKA140 and
its derivatives were used for lacZ fusion studies. Mutations
used in Figs. 5 and 6 and Table IV were introduced to the derivatives
of pKA140 by site-directed mutagenesis as described previously (4, 9).
pKA100 and its derivatives were employed for in vitro
synthesis of Inc RNA and RepZ mRNA leader for the binding assay
between them.
Isolation of Constitutive ColIb-P9 Replication Mutations from the
Measurement of ColIb-P9 Replication and repZ Expression--
The
replication ability of the In Vitro Synthesis of the RepZ Protein and the Inc
RNA--
Polypeptides directed by pCH11 or its derivative were
synthesized by the in vitro coupled
transcription-translation methods and analyzed by SDS-PAGE, followed by
autoradiography, as described previously (5). Quantitative analysis of
synthesis of the Inc RNA from pCH11 or its derivative in
vitro was conducted also as described (5).
The Binding of the Inc RNA to the repZ mRNA--
Inc RNA and
the RepZ mRNA leader were synthesized in vitro from
pKA100 or its derivative by E. coli RNA polymerase and T7 RNA polymerase, respectively, and purified as described previously (11). The purified RNAs were allowed to bind at 37 °C in a binding buffer (10 mM MgCl2, 100 mM NaCl,
20 mM Tris-HCl, pH 7.6) and analyzed by denaturing PAGE
(8.3 M urea) as described (11). Inc RNA-RepZ mRNA
hybrid was detected as a complex that persisted the gel electrophoresis
and migrated anomalously compared with free RNA species. Secondary
structures in the in vitro transcribed RNAs were analyzed
with partial ribonuclease cleavage, as described previously (10).
Isolation of Excessive ColIb-P9 Replication Mutants, Insensitive to
Inhibition by Inc RNA--
In this study, we isolated a number of
ColIb-P9 mutations affecting control of repZ with the
We employed a variety of mutant phages as starting materials in order
to obtain as many varieties of mutations as possible (Table I, columns
1-4). These parental phages generated spontaneous revertants at
various frequencies (Table I, column 5) when plated on
W3110(
The type 1 and type 2 sup mutations, isolated only from
specific rep phages, were found to derepress translation
from the repZ reading frame (Table I). Type 1 mutations,
reported previously (8), deleted C-446 or changed it to a guanine
residue (Fig. 2). These mutations strengthen the SD sequence for
repZ and weaken structure III in the context of the parental
G438A, thereby derepressing translation from the natural
repZ start codon (8). The novel type 2 sup
mutations altered C-464 to adenine and created a new AUG codon at RepZ
codon 4 (Fig. 2). Thus, the type 2 sup mutations derepressed
repZ expression by initiating translation from the AUG codon
outside of the replication control region (see under "Discussion").
Excessive ColIb-P9 Replication Mutations That Derepress Translation
from the repY Reading Frame--
Type 3 and type 4 sup
mutations fused repY in-frame with repZ (Table I
and Fig. 2) and hence are expected to derepress the repY-repZ fusion expression by impairing the inhibitory
action of Inc RNA on repY expression. Type 3 sup
mutations were single-base insertions distributed throughout
repY and were only identified in rep phages
carrying the G327A pseudoknot mutation in the loop I (Table I). Such
repY-repZ fusion mutants were isolated also from other
rep phages on W3110(
Type 4 sup mutations included more dramatic alterations
characterized by deletions of up to 23 bases and therefore occurred at
the lowest frequency of 10
First, we examined whether type 4 sup mutations impaired the
action of Inc RNA against repY translation. When the
repZ leader DNAs of three representative phages,
The type 4 sup Mutations Do Not Affect the Synthesis and Binding of
the Inc RNA--
To understand how the type 4 sup mutations
derepressed expression from the repY reading frame, we
characterized Inc RNA and RepZ mRNA encoded by
Next, we examined the effect of the type 4 mutations on the interaction
between Inc RNA and RepZ mRNA in vitro. For this
purpose, we synthesized and purified the wild-type or del2
repZ mRNA leader (293 or 288 bases, respectively) transcribed
from the NspI to Sau3AI sites depicted in Fig.
1A. When these RNAs were allowed to bind to Inc RNA as
described previously (11), they exhibited similar binding rates (Fig.
4B). The binding rate was also not affected by the
introduction of a third type 4 sup mutation W21 to the
parental 334C RNA pair (Fig. 4B). Note that these RNA pairs bound faster than the del2 or wild-type (C334A) RNA pair,
due to base changes at position 334 in both Inc RNA and RepZ mRNA (see Table I and under "Materials and Methods"). Thus, the
interaction between Inc RNA and RepZ mRNA leader depended only on
the base in the loop of structure I (at position 334), but not on bases in structure II, deleted in del2 or W21. Finally, primer
extension analyses of the Inc RNA-RepZ mRNA hybrid indicated that
the movement of reverse transcriptase on RepZ mRNA was blocked at
U-363 by Inc RNA (7) (see Fig.
5A for the position of hybrid
formation) regardless of the presence of del1 or
del2.2 These results together indicate that both
the binding rate and the final product of Inc RNA-RepZ mRNA duplex
formation were not affected by the type 4 sup mutations.
We also conducted ribonuclease cleavage experiments using the in
vitro transcribed repZ mRNA leader from
del2 (employed in Fig. 4B) and confirmed that
stem-loop II in this mutant was indeed disrupted without inducing
additional stem-loops surrounding the deleted region (data not shown).
The Type 4 sup Mutations Derepress the Translation of repY from Its
Codon 10 GUG--
At this point, a likely possibility was that the
type 4 sup mutations derepressed a cryptic start codon in
the repY reading frame that is normally sequestered in
structure II. repY codon 10 GUG could be such a start codon,
because it is located 8 bases downstream of a possible SD sequence
5'-rAGAGAU-3' at positions 391-396 (see Fig. 5A). This
hypothesis accounts for the following two contradictions, brought about
if repY(-repZ) expression from the type 4 sup mutants is initiated only from the natural
repY start codon: (i) a type 4 sup mutant was
isolated from the repY start codon mutant
To test the possibility that type 4 sup mutations derepress
translation from the repY codon 10, we altered this codon to
CUG (G405C) in pKA140del2 (Fig. 5A). As a
control, we employed the sup2044-13 mutant (inc2
G438A
It was conceivable that the del2 T379C double mutant (Fig.
5B, column 3) expressed only the shorter form of the fusion
protein, as expected for sup1060-3 and R72 mutants (see
above and Table I). To confirm this idea, we introduced G405C, which
alters codon 10 to this doble mutant. The resulting triple mutant
(G405C T379C del2) showed only 5.5
Next, we wished to examine the effect of Inc RNA on repY
expression from different mutants. To maximize its effect, we compared repY-repZ expression from isogenic pKA140 derivatives
carrying either Pinc+ or Pinc2;
inc2 reduces the amount of Inc RNA in vivo to
~18%.3 By this approach,
repY(-repZ) expression in sup2044-13
was reduced to 6% (Fig. 5C, column 7), a reduction much
larger than the value (29%) obtained by supplying the inc
gene from another plasmid (9). The same is true for control of
repZ expression; repZ expression from
inc2 was reduced to 1.4% compared with the wild-type (Table
IV, wild-type), a reduction larger than
the one previously reported, 11% (9). These results confirm that
repZ expression is reduced more efficiently than
repY expression by the same increase in the amount of Inc
RNA (9, 10). Because repY-repZ expression in del2
was increased only 1.3-fold by the introduction of inc2 (Fig. 5B, column 4), we conclude that the type 4 sup mutations retain high levels of repY-repZ
fusion expression by allowing translation initiation from the
repY codon 10. Importantly, this aberrant translation
initiation is not repressible by Inc RNA, demonstrating that the Inc
RNA binding site must be close to the repY RBS for the
normal control of repY expression by Inc RNA.
The Base Pairings in Structure II Suppress Translation from repY
Codon 10--
It was conceivable, however, that the putative start
codon mutations introduced to del2 (see Fig. 5B)
might have caused some unexpected alteration in the secondary structure
of the RNA, due to the lack of a strong RNA secondary structure
surrounding the deleted region. To overcome this problem, we attempted
to obtain point mutations in stem-loop II. We plated
To determine whether these mutations disrupted base pairing in
structure II, we introduced compensatory base-changes to pKA140-C4 (G412C) and pKA140-C9 (G405A). We found that the resulting plasmids, pKA140-C4* and pKA140-C9*, respectively, showed repZ
expression slightly lower than but comparable to their parents (Fig. 6,
C and E, left columns). When the amount of Inc
RNA transcribed from the lacZ fusion plasmids was restored
by converting their inc2 mutation to the wild-type, we still
observed high levels of repY expression in C4 and C9 (Fig.
6, B and D, right columns); however, C4* and C9*
showed repY expression reduced to 6% as compared with the
inc2 derivatives of the same strains (Fig. 6, C
and E, right columns). In addition, the repY
codon 10 mutation G405C, which disrupts the same base pair as that in
C9, did not affect repY-repZ expression in
sup2044-13 as shown above in Fig. 5C, but this
level of expression was repressible to 5% by increasing in the amount of Inc RNA (Fig. 6F). We conclude that base pairings in
structure II allow repY translation to be repressed by Inc
RNA through elimination of unregulated translation initiation from the
repY codon 10 GUG.
Furthermore, the defective control of repY by Inc RNA, as
observed in del2 (Fig. 5B), was restored when we
restored the stem-loop II structure disrupted by del2 with a
completely different sequence (Fig. 6G). Therefore, only
base pairing, not a specific RNA sequence, contributes to suppression
of this cryptic start codon.
Effect of Removing the Whole Structure II--
Finally, we
examined the effects of deleting the whole structure II, including
repY codon 10. Table IV summarizes the level of
repZ expression from pKA140 (Pinc+)
and pKA140-I2 (Pinc2) derivatives that lack the entire
structure II. When 30 nucleotides from position 404 to 433 and
encompassing structure II were deleted (in D30; see Fig. 6A
for its position), repZ expression from the
Pinc+ derivative relative to the
Pinc2 derivative was 1.8%, comparable to the ratio observed
with the wild-type (Table IV). When 32 nucleotides were deleted to fuse
repY in-frame with repZ (in D32),
repY-repZ expression from the Pinc+
derivative relative to the Pinc2 derivative was 7.6%, again
comparable to that seen with sup2044-13 (Fig.
5C). Moreover, we found that a mini-ColIb-P9 plasmid
carrying the D30 deletion was stably maintained at a copy number
1.7-fold higher than that of the wild-type (data not shown). Thus, all
of structure II could be deleted without affecting control of
repY and repZ expression by Inc RNA and
replication of the ColIb-P9 plasmid.
Inc RNA Controls Translation of repY and repZ with Different
Mechanisms--
In this study, we isolated and characterized a number
of mutations (sup) that allow excessive repZ
expression in the presence of Inc RNA (Tables I and III and Fig. 2).
Two classes of sup mutations derepressed
repY-repZ fusion expression and provided insight into how
Inc RNA represses repY expression. Type 3 sup
mutations, isolated only from the mutants altering G-327 in the loop I,
were single-base insertions at any place within repY (Table
I and Fig. 2A), and we confirmed that Inc RNA repression of
repY expression is dependent on the loop-loop interaction
between RepZ mRNA structure I and Inc RNA as proposed previously
(4, 9, 11).
Type 4 sup mutations disrupted a novel stem-loop in RepZ
mRNA (structure II) (Figs. 1B and 2), and caused
constitutive repY-repZ fusion expression by allowing
translation initiation at repY codon 10 GUG (Figs. 3-5). We
provided evidence that base pairing in structure II blocks translation
from this cryptic start codon (Fig. 6), implying that repY
codon 10 is normally sequestered in structure II. These results
indicate that structure II plays an important role in control of
repY translation by Inc RNA by blocking unregulated translation from a cryptic start codon. This is probably the only role
played by structure II, because deletion of the whole structure II
together with the cryptic codon did not significantly affect the level
and control of repZ expression (Table IV) and ColIb-P9 replication (data not shown). In agreement with this, the replication control region of the IncZ-group plasmid pMU2200, closely related to
ColIb-P9, contains the analogues of repY, inc,
structures I and III, and the pseudoknot but lacks structure II
(23).
The fact that repY translation from the natural start codon
was repressible by Inc RNA whereas that from the codon 10 GUG was not
(Fig. 5) indicates that the position of the translational start site of
repY is critical for its control by Inc RNA. Using the
replicon of a plasmid closely related to ColIb-P9 (IncB-group pMU720),
Wilson et al. (17) showed that the binding of RNA I (the Inc
analogue) failed to repress repB (the repY
analogue) translation, when a foreign nine-base sequence was inserted
between the repB SD sequence and the 3'-end of the RNA I
binding site. These findings, together with the results presented here,
establish that Inc RNA must bind in the vicinity (within 2 bases) of
the repY RBS in order to repress repY translation.
Inc RNA consists of a large stem-loop made up of 51 bases and a
single-stranded 5' leader (11). Analyses using in vitro transcribed RNAs and site-directed mutagenesis suggested that Inc RNA
binds to the repZ mRNA through two intermediate steps, a
transient interaction of its loop with the loop of RepZ mRNA structure I, followed by base pairing between 5'-end of Inc RNA and a
region proximal to the repY RBS (4, 11). Our results are
consistent with the model that repY translation is inhibited due to the latter step. Similar models were proposed for antisesne RNA-mediated inhibition of leader peptide synthesis in the IncFII R1
(12, 15, 24) and IncB pMU720 (17, 25) plasmids. It is noteworthy that
Inc RNA controls repY expression with a mechanism quite
different from that revealed for repZ expression,
i.e. inhibition of the pseudoknot formation by the transient
loop-loop interaction (see the Introduction). Based on our previous and
present analyses, it could be proposed that two different parts of Inc
RNA, the loop and 5' leader regions, directly inhibit RNA-RNA
interactions critical for repZ and repY
expression, respectively.
Effect of the Translating Ribosome on mRNA Translation--
We
have identified two GUG start codons in the coding region of
repY; one (repY codon 10) is silenced by
structure II, and the other is located in structure III, which serves
as the repZ start codon (Fig. 5A). Translation
initiation from the latter requires both the termination event of
repY translation and the complementarity between 5'-side
stem of structure III and the loop of structure I for the pseudoknot
formation (8-10). Yet, in the case of the MS2 RNA phage, a simple
passage of ribosome through the coat gene region is sufficient to open
the RBS for replicase and lysis proteins (26).
What determines the accessibility of a certain RBS located in the
coding region during the process of translation? It is likely that the
balance between the affinity of the ribosome to each RBS and the
stability of the secondary structure that blocks it make up this
difference. In the case of MS2, the replicase and lysis protein RBSs
are blocked with weak (bulged-out) secondary structures (26). Thus, the
passage of the ribosome through the coat gene region may be sufficient
to trigger ribosome binding to these RBSs and block the formation of
RNA secondary structures. In the case of repY codon 10, structure II may refold immediately, and preclude ribosome binding to a
relatively weak RBS. In the case of repZ start codon, the
repZ RBS is too weak (8) to stimulate ribosome binding
during passage of a translating ribosome. Instead, a pseudoknot is
induced during the translational termination of repY and
serves to keep the repZ RBS accessible for a long enough time to allow ribosome-RBS interaction. Kinetic analyses of mRNA structure formation, such as developed by Ma et al. (27), in combination with coupled protein synthesis, will be important to test
these models.
Importance of mRNA Secondary Structures in Translational
Control of repZ--
The identification of a second inhibitory
structure (II) also suggests the importance of mRNA structure in
the translational control of repZ. This idea is consistent
with the previous characterization of structure III with the
sup mutations classified here as type 1 (8) and the
isolation of the type 2 sup mutations locating outside of
the replication control region (Tables I and III and Fig.
2A). Interestingly, the isolation of both type 1 and type 2 mutations appears to require the parental mutations that partially disrupt structure III and creating a stronger SD sequence near the
start codon used (8).3 Thus, we propose that a weak RBS is
critical for inhibition of translation by mRNA secondary structures
and essential in maintaining the integrity of ColIb-P9 replication control.
A wide variety of translational control mechanisms using mRNA
structures evolved in prokaryotes (2), probably because the formation
of secondary structures sequestering RBS can directly compete with
ribosome binding (28, 29). In contrast, eukarytic ribosomes scan for
the first AUG with the help of numerous initiation factors, as they
melt secondary structures found in the 5' leader of mRNA (30, 31).
Accordingly, translational control in eukaryotes occurs by a variety of
mechanisms that modulate the activity of initiation factors
(32-35).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
group)
depends on expression of the replication initiator protein RepZ,
encoded by its autonomous replication region (5), shown schematically
in Fig. 1A. The level of repZ expression is
strictly controlled at the translational level by the actions of
positive and negative regulatory elements, repY (6) and
inc (5, 7), respectively. repY is a short open
reading frame encoding 29 amino acids, the 3'-end of which overlaps
with the 5'-end of repZ (6) (see Fig. 1A). An
amber mutation of repY codon 11 (rep57) did not
allow repZ to be translated, indicating that repY
translation is required for repZ translation (6). The
process of repY translation and its termination, rather than
the RepY polypeptide, is required for the induction of repZ
translation (8). inc is situated 5' to repY and
encodes the antisense Inc RNA with a size of ~70 bases (5, 7) (see
Fig. 1A). Inc RNA represses repZ expression by
binding to a complementary region within the repZ mRNA
(7).
plasmid
ColIb-P9 and its close relatives in IncB group (17) provide unique
opportunities to study a single antisense RNA that controls
expression of two different genes.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ind
)
were used as the hosts of
:ColIb-P9 hybrid phages. Strain MC1061 (lacX74) (18) was employed for lacZ fusion
studies and as the host for mini-ColIb-P9 plasmids.
CH10 (5) was a hybrid between
VIII (cIam) (19) and the
3.0-kilobase EcoRI fragment of ColIb-P9 (5) (Fig.
1A) that shows autonomous replication.
CH10-1 and
CH10-2 are derivatives of
CH10 carrying the Inc-promoter
mutations inc1 (A400C) and inc2 (T374C),
respectively (5). The hybrid phages employed for isolating
inc-insensitive replication mutants are listed and described
in Table I, columns 1-4. Because we
realized that the original
CH10 contained a moderate copy-up
mutation altering C-334 to A (4, 11),
CH10W with a cytosine residue
at position 334 was also employed for isolating the
inc-insensitive mutants. As C334A had only minor effects on
the replication control of ColIb-P9 (4, 9-11) and
CH10 has served
as the useful source of a variety of replication control mutations
(5-9), we describe
CH10 as wild-type in this study and distinguish
it from the 334C wild-type carried on
CH10W.
CH10sup57-1 (6) and
2sup2044-13 (9) were
pseudorevertants, isolated previously by plating
CH10rep57 and
2rep2044, respectively, on
W3110(
ind
).
Isolation of excessive ColIb-P9 replication mutations, insensitive to
the action of Inc RNA
CH10,
CH10W, or
CH10 rep hybrid phage (described in columns 1-4) was
plated on W3110(
ind
) cells harboring pCH11
(Inc+) (or pCH11W (Inc+, 334C) for
CH10W). The
sequences of altered start codons in repY or repZ
are given in parentheses in column 4. The frequency of spontaneous
reversion (sup mutations) for each phage is given in column
5. We isolated and plaque-purified three or four such revertants from
each parent and sequenced them to determine the site of mutation as
described previously (9). These revertants, except for those isolated
from
2rep2006 (and its isogenic mutants) and
2rep2044, were found to contain identical or similar
mutations in at least two independent isolations. Therefore we listed a
single representative sup mutant (columns 6 and 7). The
revertants were classified into four types, as shown in column 10, according to whether or not they carried repY-repZ fusion
mutations (column 8), or disrupted stem-loop II (column 9).
2rep2006 (inc2 G327A) generated two groups of
revertants showing different burst sizes on
W3110(
ind
)/pCH11, one with that of ~30, and
the other with that of >80. The former, consisting of type 3 mutations, is isolated more frequently than the latter consisting of
type 4 mutations.
1rep1030 (inc1 G327A)
generated type 3 mutations more frequently (column 5), because
C403
insertion occurred predominantly after four consecutive cytosine
residues, including one altered by inc1 at position 400. Because
2rep2044 (inc2 G438A) generated both
type 1 and type 4 mutations in a similar frequency, we isolated and
sequenced ~30 revertants in a single isolation, and part (this table)
or all (Fig. 2B) of the isolated sup mutations
are reported.
List of plasmids used in this study
:ColIb-P9 Hybrid Phages--
The method of isolation of ColIb-P9
replication control mutations using the
:ColIb-P9 hybrid phage
system was described previously (5-9). In this study, we isolated
ColIb-P9 mutations that show excessive replication in the presence of
the inc gene in trans. We employed a wide variety
of the
:ColIb-P9 species as parents (Table I, columns 1-4). The
parental phages included
CH10,
CH10W (334C wild-type), and
CH10 rep phages defective in the pseudoknot formation or
the repY or repZ start codon, thereby reducing
repZ expression. These phages did not form visible plaques
on W3110(
ind
) cells, because the
replication from the
portion was inhibited by the
repressor
encoded by the
ind
prophage, and the
replication frequency from the ColIb-P9 portion was not sufficient to
support the lytic growth of the hybrid phage. To eliminate possible
revertants, the replication of which was repressible by Inc RNA, we
plated these phages on W3110(
ind
) harboring
pCH11 (inc) (or pCH11W for
CH10W). In this condition, they generated visible plaques at frequencies between 10
5
and 10
9 (Table I, column 5), a value significantly higher
than the frequency (10
10) at which the
VIII vector
generated visible plaques. Such revertants were purified to homogeneity
and analyzed for sequencing the entire replication control region of
ColIb-P9 as described (9). In most cases, we located additional
mutations in the control region that were responsible for increasing
repZ expression, and designated them as sup
mutations. In rare occasions, however, we did not find any mutation in
the ColIb-P9 control region. This was because mutations occurred in the
portion, as confirmed by the analysis of the
VIII vector excised
from the revertant phage DNA (5).
C phages, listed in Fig.
6A, were isolated as pseudorevertants by plating
2sup2044-13 on W3110(
ind
)
harboring pCH11.
:ColIb-P9 hybrid phages was determined by
one-step growth on W3110(
ind
) cells as
described previously (5). The burst size was calculated by the number
of progeny phages divided by the number of adsorbed phages. The level
of repZ expression was estimated by measuring the
-galactosidase activity expressed from MC1061 (Lac
)
carrying pKA140 or its derivative (9). The specific activity of the
enzyme was expressed as Miller's units (22). The method of
determination of the copy number (4, 11) of mini-ColIb-P9 plasmids was
described previously. The values reported in this study are averages
from at least three independent experiments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
:ColIb-P9 hybrid phage system. After initial characterization of
potentially interesting mutants, we excised the mutant phage DNA
corresponding to the replication control region (depicted in Fig.
1A) and subcloned it into
different vectors for further characterization (see under "Materials
and Methods"). With the
:ColIb-P9 hybrid phage system, we expected to obtain mutations that cause a large increase in repZ
expression, even though they did not support the vegetative replication
of ColIb-P9 (4, 5).
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Fig. 1.
Control of replication of plasmid ColIb-P9
involving antisense RNA and the repZ mRNA
pseudoknot. A, the autonomous replication segment of
ColIb-P9. Boxes denote the functional segments
inc, repY, repZ, CIS, and
ori. RepZ is presumed to act on ori and stimulate
the initiation of unidirectional replication (dotted arrow)
from the G-site (36). Transcription termination of RepZ
mRNA at CIS is additionally required for ColIb-P9
replication (37). ter denotes the replication termination
site. Short, thick arrow represents Inc RNA. Open
and closed circles on the transcripts represent the
5'-rGCC-3' and 5'-rGGC-3' sequences, respectively, critical for the
positive (+) and negative ( ) control of repZ translation.
The recognition sites for restriction enzymes used in this study are
shown by arrowheads; Bs, BstPI;
E, EcoRI; E(B), EcoRI site
converted from the original BglII site; H,
HincII; N, Nsp(7524)I; S,
SalI; Sa, Sau3AI. B,
nucleotide sequence and secondary structures in RepZ mRNA leader
region. Roman numerals indicate secondary structures
identified in the RepZ mRNA in vitro (10). The SD
sequences for repY and repZ are shown by
asterisks. Start codons of repY and
repZ are underlined. The stop codon of
repY is double-underlined. The two largest
complementary sequences are boxed. The bases critical for
the pseudoknot formation are shown by brackets. Nucleotide
numbers are coordinated from the BglII site at the left end
of the replication region. C, the proposed regulatory model
of repZ expression. Panels a, b, c, and
d represent the possible states of RepZ mRNA.
Roman numerals indicate stem-loop structures. The region
complementary to Inc RNA is bracketed. Open boxes
represent the repZ RBS. Filled boxes represent
the complementary sequences for the pseudoknot formation.
Filled and open circles represent start and stop
codons for repY, respectively. See the Introduction for
details on this model. Panel a depicts the nascent RepZ
mRNA. Panel b describes conformational changes induced by
repY translation. Panels c and d
describe the steps of Inc RNA binding.
ind
)/pCH11, an E. coli
strain expressing the wild-type Inc RNA. The spontaneous revertants
identified showed burst sizes of >30, which is typical of lytic phages
(data not shown) and is consistent with the growth phenotype. They were
found to contain mutations (designated here as sup) in the
repZ leader region, as described in Fig.
2 and Table I, columns 6-9, and
classified into four types according to the nature of the mutations
(Table I, column 10). These sup mutants exhibited
repZ expression more than 100 times higher than their
parents, when examined using a translational lacZ fusion
(see under "Materials and Methods"). A subset of the results is
summarized in Table III. These high
expression levels were not affected by the presence of the
inc gene in trans, as was expected from the
mutant selection scheme (data not shown). Thus, all the identified
mutations derepress repZ expression regardless of the Inc
RNA inhibitory function.
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Fig. 2.
Locations of sup mutations
that derepress repY or repZ
expression in the repZ leader region.
A, locations of the sup mutations, listed in
Table I except ones from 2rep2044, are indicated on the
nucleotide sequence of the repZ leader region, numbered as
in Fig. 1. Arrowheads indicate base substitutions or
single-base deletions or insertions. Thick horizontal bars
denote the positions of deletions. Amino acid sequences for
repY and repZ are shown below the nucleotide
sequence. The start codons for repY and repZ are
shown in boldface. Asterisks denote the proposed
SD sequences. Positions of stem-loop structures II and III are
indicated by horizontal arrows. Underlined are
the
10 and
35 sequences for the Inc RNA promoter in the
complementary strand. B, locations of all the sup
mutations isolated from
2rep2044 are described as in
A. Note that T-374 and G-438 are altered to C and A
(boldfaced) in the nucleotide sequence, due to the parental
mutations inc2 and G438A, respectively.
:ColIb-P9 replication and repZ expression in the excessive
replication mutants, insensitive to inhibition by Inc RNA
:ColIb-P9 sup mutants and
their parents were measured on W3110(
ind
) as
described previously (5) and shown below. To measure the level of
repZ expression, pKA140 derivatives carrying the
sup mutations were constructed and introduced into MC1061
(Lac
) for determination of
-galactosidase activity. The
value in Miller's unit (22) is presented here as RepZ activity.
ind
), but
were not able to grow on W3110(
ind
)/pCH11
(inc) (6, 9). We previously showed that the wild-type Inc
RNA binds RepZ mRNA carrying G327A >50-fold more slowly than it
binds the wild-type RepZ mRNA, due to the C:A mismatch at position 327 (11). Therefore, the isolation of type 3 mutations from the G327A
mutants reinforces the idea that Inc RNA represses repY expression in a manner requiring the loop-loop interaction with structure I.
9 (Table I). Interestingly,
these mutations commonly disrupted a part of stem-loop structure II
(Fig. 2). In contrast to other sup mutations, the type 4 mutations were isolated from a variety of
:ColIb-P9 hybrid phages.
Thus, disruption of structure II alone appears to be sufficient for the
lytic growth of
:ColIb-P9. Because involvement of structure II is
novel, we further characterized these mutations.
CH10del1,
CH10del2, and
CH10del57, were subcloned into pBR322 and examined for
in vitro protein synthesis, we observed polypeptides (Fig.
3, lanes 5, 6, and
9, respectively, arrowheads), slightly smaller
than the RepY-RepZ' fusion protein (29 kDa) (lane 3) but
significantly larger than the RepZ' protein (26 kDa) (open
triangles) from the wild-type or inc1 (lane 1, 2, or 8). Because translation from the natural
repZ start codon was not observed, type 4 sup
mutations bypassed the control of repY expression by Inc
RNA. However, it is not clear by this analysis whether the
repY-repZ fusion proteins observed in the type 4 sup mutants were synthesized exclusively from the natural
repY start codon (see below).
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Fig. 3.
In vitro protein synthesis from
three type 4 sup mutants, del1,
del2, and del57. The
35S-labeled proteins synthesized from pCH11 and its mutant
derivatives were separated by 7.5% SDS-PAGE (see under "Materials
and Methods"). Arrowheads and open triangles
indicate the repY-repZ fusion and repZ proteins,
respectively. Ovalbumin (45 kDa) and carbonic anhydrase (31 kDa) were
used as the size makers. Mutations in the plasmids are listed across
the top. Lane 8, pBR322 was used instead of pCH11
plasmids.
CH10del1 and
CH10del2. In vitro RNA
synthesis analyses indicated that del1 and del2
did not reduce the amount of Inc RNA synthesized from the corresponding
derivatives of pCH11 (Fig. 4A,
lanes 5 and 6), as compared with Inc-promoter mutations inc1 and inc2 (lanes 3 and
4). Northern blot analysis using RNAs isolated from
W3110(
ind
) carrying pCH11del1
and pCH11del2 confirmed that they produced Inc RNA and RepZ'
mRNA in vivo in amounts comparable to those produced
from the wild-type pCH11.2
Together, these results indicate that at least the del1 and
del2 mutations derepress repY(-repZ)
expression at the translational level without affecting Inc RNA
synthesis.
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Fig. 4.
Type 4 sup mutations do not
affect the production and binding of Inc RNA. A, RNAs
synthesized from pCH11del1 and pCH11del2.
Transcripts synthesized in vitro from pCH11 and its mutant
derivatives were labeled with [ -32P]UTP and separated
by 3% PAGE in the presence of 8.3 M urea. RNA I, encoded
by the pBR322 vector portion, was used as the internal standard. The
DNA fragments prepared by digesting pBR322 with HaeIII
restriction enzyme were used as size markers (in base pairs). Mutations
in the plasmids are listed across the top. Lane
7, pBR322. B, binding of Inc RNA to the RepZ mRNA
leader carrying type 4 sup mutations. 10 nM
unlabeled RepZ mRNA leader carrying del2
(
5(421-425)) or W21 (
11(421-431)) was incubated with 1 nM 32P-labeled cognate Inc RNA. Parental RepZ
mRNA leaders (wild-type for del2 and
334C for W21) were used as controls. Aliquots withdrawn at
times indicated were analyzed by denaturing PAGE, followed by
phosphorimaging analyses with a Fuji BAS2000 BioImage analyzer. Ratio
of free to total Inc RNA was calculated and plotted against time.
Wild-type RNAs bound more slowly than did 334C RNAs, due to the
alteration of a base (at position 334) in the lower Inc loop region
(11).
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Fig. 5.
Translation initiation from the
repY codon 10 GUG is not repressed by Inc RNA.
A, the 5'-terminal region of repY and mutations
employed in this analysis. The nucleotide sequence of the RepZ mRNA
from positions 350 to 430 is depicted. Mutations used are indicated by
vertical arrows. The thick bar indicates the
position of deletion in del2. The 10 and
35 sequences
for the Inc RNA promoter in the complementary strand are
underlined: inc2 reduces the synthesis of Inc
RNA. Codons 1 and 10 of repY are shown in
boldface. The proposed SD sequences for these codons are
shown by asterisks. Horizontal arrows denote the
position of stem-loop structure II. The 5'-terminal sequence of Inc RNA
is shown above the RepZ mRNA sequence at its binding site in
parentheses. The amino acid sequences predicted for repY and
its truncated reading frame found in del2 are shown below
the nucleotide sequence. B and C, effect of
altering different repY start sites in del2
(B) and sup2044-13 (C). Different
repY start site mutations, as shown in A and
indicated below each column, are introduced to pKA140del2
(Pinc+ del2, column 1 in
B) or pKA140-I2sup2044-13 (Pinc2 G438A
T454, column 1 in C), encoding
repY-repZ-lacZ fusion.
-Galactosidase activities from
MC1061 (Lac
) carrying the resultant plasmids are shown in
Miller's units (22) as the level of repY(-repZ
fusion) expression. Percentages of expression relative to the value
from the parental plasmid (column 1 in each panel) are
indicated. Bars above each value represent S.D. from at
least three independent measurements.
1rep1060 (Table I); and (ii) a four-base deletion in
R72 disrupted a part of structure II, but did not fuse
repY in-frame with repZ, and hence can be
classified as a subtype of type 4, type 4' (Table I and Fig.
2B). The
R72 mutation may derepress repZ
translation from the GUG codon at positions 410-412 (in-frame with
repZ) by bringing it closer to the presumed SD sequence
(positions 391-396) for the repY codon 10.
T454) that produces only RepY-RepZ fusion (and not RepZ)
protein as described previously (9). We found that changing
repY codon 10 (G405C) in the del2 mutant reduced
the level of repY-repZ expression to 1.6% (Fig. 5B,
column 2), whereas it did not alter repY-repZ
expression in sup2044-13 (Fig. 5C, column 2).
Conversely, altering the natural repY start codon to ACG
(T379C) in del2 reduced repY-repZ expression to
66% (Fig. 5B, column 3), whereas the same mutation reduced
expression in sup2044-13 to 2.1% (Fig. 5C, column
3). In addition, altering each of three bases of the proposed
repY SD sequence (5'-rGGGU-3') at positions 366-369 (Fig.
5A) reduced repY expression to 51-74% in
sup2044-13 (Fig. 5C, columns 4-6). These results not only
confirm the location of the repY RBS but also indicate that
the translation of the RepY-RepZ fusion protein in del2 is
initiated mainly from the repY codon 10.
-galactosidase units
of repY expression, a value 320-fold lower than that of
del2 T379C, and even 8-fold lower than the level from
del2 G405C. Thus, the RepY-RepZ fusion protein from the
del2 T379C double mutant is initiated only from the
repY codon 10.
The effect of deletion of the whole structure II on control of repZ
expression
) cells carrying
these plasmids were grown to A600 = ~0.4, and
tested for
-galactosidase activity as described previously (9). The
values in Miller's units (22) are presented under Pinc2 and
Pinc+ as RepZ activity. The percentage of activity
from the Pinc+ derivative relative to the
Pinc2 derivative is given under
Pinc+/Pinc2.
2sup2044-13 (repY-repZ fusion) on
W3110(
ind
)/pCH11, and isolated clear-plaque
forming mutants. As shown in Fig.
6A, we were able to isolate
mutations that were presumed to interrupt structure II. Among eight
independent mutations, two altered G-412 to T (G412T), three changed
G-412 to C (G412C), two changed G-425 to A, and still another had G-405
altered to A (G405A). Note that the last mutation altered the
repY codon 10 to a typical AUG start codon. We constructed
pKA140-I2sup2044-13 derivatives, transformed MC1061
(Lac
) in order to assay their
-galactosidase activity
and found the
-galactosidase levels 2-fold higher than that from
sup2044-13 (data not shown; see also Fig. 6, B
and D, left columns).
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Fig. 6.
The repY codon 10 GUG is
sequestered in the stem-loop structure II. A, isolation
of point mutations in structure II. Deduced RepZ mRNA sequence from
position 400 to 435 is shown with the stem-loop II structure. The
repY codon 10 is boxed. Point mutations found in
C phages (indicated in parentheses), isolated from
2sup2044-13 (inc2 G438A
T454), are shown by
arrows and altered bases. Bars below the sequence
denote positions of deletions used in Table IV. B-E, the
structures of stem-loop II from C4, C9, and their derivatives deduced
with the program of Zuker and Stiegler (38) are shown with altered
bases in boldface. Histograms show the levels of
repY(-repZ fusion) expression from these mutants
(left columns) and their derivatives carrying
Pinc+ (right columns), measured with
the lacZ fusion method (see under "Materials and
Methods"). Bars above each
-galactosidase value
represent S.D. from three independent measurements. Percentages of
expression of the Pinc+ derivatives are also
indicated. F and G, G405C and a six-base
insertion were introduced to pKA140-I2sup2044-13 and
pKA140del2, respectively. repY expression from
their Pinc2 and Pinc+ derivatives is
compared as described above for B-E. The 6-base insertion
was introduced to pKA140del2 by replacing the 9-base
sequence following the repY codon 10 with the 15-base
sequence 5'-rUAUUGCAGUGUAUGC-3', without altering the deletion junction
found in del2. Bases different from those in wild-type
structure II are shown in boldface. The value in the
left column in F is taken from column
2 in Fig. 5C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are indebted to Jim Anderson for critical reading of the manuscript and to Kiyotaka Shiba for discussion and sharing the results prior to publication. K. A. thanks Alan Hinnebusch for his understanding and discussion.
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FOOTNOTES |
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* This work was supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture (Monbusho) of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a Japan Society for Promotion of Science Fellowship for Japanese Junior Scientists. To whom correspondence should be addressed: Bldg. 6A, Rm. B1-A13, Laboratory of Eukaryotic Gene Regulation, NICHD, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-594-7240; Fax: 301-496-8576; E-mail: kasano{at}aghmac1.nichd.nih.gov.
¶ Current address: Division of Molecular Genetics, National Institute of Neuroscience NCNP, Kodaira, Tokyo 187, Japan.
2 K. Shiba and K. Mizobuchi, unpublished observations.
3 K. Asano and K. Mizobuchi, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: SD, Shine-Dalgarno; RBS, ribosome binding site; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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