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J Biol Chem, Vol. 275, Issue 2, 1269-1274, January 14, 2000
From the The antisense Inc RNA encoded by the IncI Antisense RNAs bind to the complementary regions (or
"sense" strands) of their target RNAs and exert a variety of
regulatory functions (1, 2). Some of them are encoded in the autonomous replication regions of plasmids and negatively control their
replication and copy number (3). The copy number of the plasmid is
proportional to the intracellular concentration of its antisense RNA,
which, in turn, down-regulates the frequency of plasmid replication, thereby establishing a negative feedback loop for the maintenance of
constant copy number. These antisense RNAs, when expressed in a
bacterial cell, repress the replication of identical or closely related
plasmids introduced by means of transfer or transformation. This
phenomenon is called incompatibility
(Inc)1 and is used to
classify plasmids: i.e. if two different plasmids are
incompatible in a single bacterial cell, they belong to the same Inc
group (4).
There are essentially two types of antisense RNAs that show the highest
binding rate constants of ~106/M/s (1). High
copy number plasmids including pMB1 and ColE1 encode one type
(designated here as class H for high copy) of such antisense RNAs.
These antisense RNAs have the size of ~100 bases, form three
stem-loops with a single-stranded 5' leader, and bind to the preprimer
RNAs for the leading strand DNA synthesis, thereby inhibiting formation
of mature primer RNAs (1). The other type of rapid binders (class L for
low copy) are encoded by low-copy-number plasmids including the members
of IncFII, IncI It is established that the process of binding of antisense RNA I (A) to
its target RNA II (T), encoded by ColE1 (class H), comprises a series
of reactions producing more stable intermediates,
The C* complex formed in class L was characterized only in the
IncI
Structural Analysis of Late Intermediate Complex Formed
between Plasmid ColIb-P9 Inc RNA and Its Target RNA
HOW DOES A SINGLE ANTISENSE RNA REPRESS TRANSLATION OF
TWO GENES AT DIFFERENT RATES?*
§ and Department of Biophysics and
Biochemistry, Graduate School of Science, University of Tokyo,
Hongo, Tokyo 113-0033, Japan and the ¶ Department of Applied
Physics and Chemistry, University of Electro-Communications,
Chofu-shi, Tokyo 182-8585, Japan
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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ColIb-P9 plasmid replicon controls the translation of repZ
encoding the replication initiator and its leader peptide
repY at different rates with different mechanisms. The
initial loop-loop base pairing between Inc RNA and the target in the
repZ mRNA leader inhibits formation of a pseudoknot
required for repZ translation. A subsequent base pairing at
the 5' leader of Inc RNA blocks repY translation. To delineate the molecular basis for the differential control, we analyzed
the intermediate complexes formed between RepZ mRNA and Inc
RNA54, a 5'-truncated Inc RNA derivative. We found that the initial base pairing at the loops transforms into a more stable intermediate complex by its propagation in both directions. The resulting extensive base pairing indicates that the inhibition of the
pseudoknot formation is established at this stage. Furthermore, the
region of extensive base pairing includes bases different in related
plasmids showing different incompatibility. Thus, the observed
extensive base pairing is important for determining the incompatibility
of the low-copy-number plasmids. We discuss the evolution of
replication control systems found in IncI, IncB, and IncFII group plasmids.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(IncI1) and IncB groups (1, 2). These
antisense RNAs are ~70-100-base long, fold essentially into a single
large stem-loop of ~50 bases, and control the expression of the
plasmid replication initiator (rep) genes at the
translational level. Specifically, they block the translation of a
rep leader peptide (RLP) coupled to the translation of
rep (5-9), by binding to the vicinity of the
ribosome-binding site (RBS) for the RLP (10-12). In the case of
IncI ColIb-P9 and IncB pMU720 plasmids, the translation of RLP
(repY and repB) induces formation of a pseudoknot
between the target stem-loop and a sequence preceding the
rep (repZ and repA, respectively) RBS,
which is required for rep translation (13-15). The
antisense RNA additionally inhibits the pseudoknot formation by binding
to the loop of the target stem-loop structure (16). Because of the
different mechanisms, the antisense RNA represses rep
expression more efficiently than RLP expression, thereby establishing
the constant level of Rep protein and hence the plasmid copy number
(16). We have been studying this unique differential control exerted by
the antisense Inc RNA of the ColIb-P9 plasmid.
where Cs is the stable final product, C** is
characterized by its altered RNase sensitivity and C* is the unstable
complex, the formation of which is inferred from the inhibition
kinetics. The C* complex appears to consist of multiple contacts at
loops and 5'-single stranded leader, with the inhibitory dissociation constant (Ki) of 11 nM (17, 18). In the
C** complex, base pairing in each loop-loop interaction may stack on
the stems of the antisense and target RNAs, which altogether provides
the equilibrium dissociation constant (Kd) of 0.1 nM (18). Different incompatibilities among ColE1-type
plasmids (class H) can be explained at the level of either C* or
C** formation (17, 18).
(Eq. 1)
ColIb-P9 system: Formation of C* is mediated by base pairing at
the 5'-rGGCGG-3' sequence in the target loop (Fig. 1, step f), and shows a
Ki of 6-10 nM (16). It is proposed that
this initial interaction is stimulated by a specific loop structure
conferred by the target loop sequence, 5'-rUUGGCG-3' (Fig. 1,
step e) (16, 19), which is conserved in the target loop for
class L antisense RNAs (20, 21). Formation of C* can directly compete
with the pseudoknot required for ColIb-P9 repZ translation,
explaining how the antisense Inc RNA represses repZ
translation at the level of loop-loop interaction (Fig. 1, steps
b and f) (16).
View larger version (18K):
[in a new window]
Fig. 1.
Pathway for antisense Inc RNA pairing and
activator pseudoknot formation in the control of Colb-P9
replication. Nascent RepZ mRNA (step d) undergoes
different conformational changes for positive (steps a-c)
and negative (steps e-i) control. Position of
repZ and repY is shown by thin arrows
in step d. SD(Y) and SD(Z) indicate
the Shine-Dalgarno sequences for repY and repZ,
respectively, and are shown as struck through when they are
masked. Closed and open boxes denote the critical
5'-rGGC-3' and 5'-rGCC-3' sequences, respectively. Bars
between the lines (RepZ mRNA or Inc RNA) represent pseudoknot or
intermolecular base pairings. Thick arrows indicate the
status of repY or repZ translation in each step.
Step g of Inc RNA pairing, the major focus of this study, is
highlighted by a dotted box.
No detectable incompatibility between IncI and IncFII plasmids (22)
can be explained at the level of C* formation, because one of the bases
critical for C* formation in IncI ColIb-P9 is altered in IncFII
plasmids (16). Despite sharing the bases critical for C* formation,
IncI and IncB plasmids are compatible for several generations
although they show weak incompatibility thereafter (22). Therefore, the
reduced stability of the complex formed later than C*, most likely C**,
must be responsible for the observed compatibility between IncI and
IncB plasmids.
In this report, we focus on the secondary structure of a late
intermediate complex (C**) formed between Inc RNA and its target, encoded by IncI ColIb-P9 (highlighted in Fig. 1, step g).
We find that the initial transient interaction (C*) between RepZ mRNA and Inc RNA54, a derivative of Inc RNA lacking its
5'-single stranded leader, transforms into a more stable complex
altering the sensitivity of the target RNA region to a variety of
RNases. Our results show that the region of extensive base pairing
encompasses the bases different from the IncB plasmids, consistent with
the idea that C** is an important determinant of plasmid
incompatibility. Furthermore, we find that the extensive intermolecluar
base pairing is separated at the site of initial base pairing,
suggesting a stacking trend between each intermolecular helix and the
stem of Inc RNA or its target. This unusual structure at the late
intermediate step provides insights into the molecular basis for the
unique differential control by Inc RNA.
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RNA Preparation-- Preparation of Inc RNA, Rep RNA293 (19), Inc RNA54, Ps14, and SI54 (16) was described previously. Inc21 (5'-GUCGUUCCGCCAAGUUCGUAA-3') was chemically synthesized and purified as described (16).
Binding of Inc RNA to RepZ mRNA in Vitro-- Binding of Inc RNA to RepZ mRNA was conducted in the binding buffer (50 µl) containing 10 mM MgCl2, 100 mM NaCl, and 20 mM Tris-HCl (pH 7.6) at 37 °C and analyzed by denaturing polyacrylamide gel electrophoresis in the presence of 8.3 M urea (16, 19). Inc RNA-Rep RNA hybrid was detected as a complex that was stable during the denaturing gel electrophoresis and migrated anomalously compared with free RNA species.
Secondary Structural Analyses on the Late Complexes between Rep
RNA293 and Inc RNA54 or
Ps14--
40 nM 5' 32P-Rep
RNA293 (14) was incubated with indicated amounts of Inc
RNA54 and Ps14 in 5 µl of binding buffer (19)
containing 0.25 µg of yeast tRNAs at 37 °C for 30 min and digested
partially with 1 µl of appropriately diluted RNases at 37 °C for
10 min. The reaction was terminated by adding 5 µl of stop solution
(19) on ice. 4 µl of the sample was resolved by 8% polyacrylamide
gel electrophoresis (8.3 M urea). Gel was dried for
autoradiography or phosphorimaging analysis with the Fuji BAS2000
BioImage analyzer. For the measurement of Kd, 0, 25, 50, 100, and 400 nM Inc RNA54 or
Ps14 was employed for the cleavage experiments as above,
and the amount of RNase T1 products cleaved at G-327 and G-328 was
quantitated together with the cleavage product at G-360 as an internal
standard. Kd was calculated as [Inc
RNA54 or Ps14]y=1/2 
20 nM, where y is the relative intensity of the
G-327 and G-328 bands.
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Inc RNA54 Binds to Stem-Loop I in Two Steps--
We
previously synthesized three RNA species, Inc RNA54,
Ps14, and SI54 (see under "Experimental
Procedures") and examined their effects on the interaction between
Inc RNA and Rep RNA293, a 293-base RNA corresponding to the
repZ mRNA leader (16). Inc RNA54 is an Inc
RNA derivative lacking the 5' single-stranded leader and did not form a
stable complex with Rep RNA293 under the denaturing gel
conditions. Ps14 is a single-stranded 14-base portion of
RepZ mRNA with a 7-base sequence complementary to loop I, and its
binding to the target stem-loop (designated structure I) mimics the
repZ mRNA pseudoknot. SI54 is a 54-base RNA
corresponding to the stem-loop I of RepZ mRNA. Fig.
2 shows a reproduced result of the
kinetic analyses on the inhibition of Inc RNA binding to Rep
RNA293 by these three inhibitory RNAs. As shown by the
filled squares in Fig. 2, A and B,
32P labeled Rep RNA293 or Inc RNA reduced the
fraction of unhybridized species, due to its hybridization to excess
unlabeled Inc RNA or Rep RNA293, respectively. As shown by
open symbols in Fig. 2, A and B, the
presence of inhibitory RNAs decreased the initial rate of the
hybridization with Ki values of 6-10
nM, as reported previously (16). Thus, the three RNAs
formed transient, reversible complexes with the partners, and competed
with the rate-limiting step of the hybridization reaction (complex C*; see the second steps in Fig. 2, C and E).
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During the course of these analyses, we realized that the binding between Inc RNA and Rep RNA293 was significantly retarded within 2 or 3 min in the presence of Inc RNA54 and SI54, carrying a large stem-loop structure (Fig. 2, A and B). Because Inc RNA54 and SI54 are present in large excess over 32P-Rep RNA293 and 32P-Inc RNA, respectively, we hypothesized that this secondary inhibition is due to their rapid, irreversible formation of a different complex with the complementary region in the 32P-Rep RNA293 or 32P-Inc RNA. We presumed that such a complex did not withstand the denaturing gel electrophoresis for the detection of Inc RNA-Rep RNA hybrid (see the third steps in Fig. 2, C and E). In contrast, the secondary inhibition was not observed with the single-stranded Ps14 RNA in this time frame (Fig. 2A). However, the preincubation (>15 min) of excess Ps14 (400 nM) with 32P-Rep RNA293 completely blocked Inc RNA binding (data not shown). Thus, the initial base pairing with Ps14 may transform into an irreversible complex more slowly than the complex with Inc RNA54 (Fig. 2D).
Secondary Structure Analyses of the Late Complex between RepZ
mRNA and Ps14--
To examine the possibility that the
initial base pairing (C*) between Inc RNA and its target transforms
into an irreversible intermediate interaction corresponding to C**, we
analyzed the secondary structure of 5'-32P labeled Rep
RNA293 using RNases V1 (specific to double-stranded RNA),
T1, U2, PhyM, and CL3 (specific to an unpaired G, A, U/A, or C residue,
respectively), after the preincubation (30 min) of 32P-Rep
RNA293 with Inc RNA54 or Ps14. Fig.
3 shows the gel electrophoretic patterns
of the cleaved products of Rep RNA293. Fig.
4 describes the deduced secondary
structure of the target stem-loop I in RepZ mRNA. Cleavage
experiments with 32P-Rep RNA293 alone (Fig. 3,
lanes 1, 7, 13, 16, and 20) confirm the
previously proposed structure of stem-loop I (14, 19), as shown in Fig.
4A. G-327 and G-328 at the top of the loop were strongly
cleaved by RNase T1. These two bases are most critical for both the
pseudoknot formation (13, 14) and Inc RNA binding (19).
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In the presence of Ps14, the structure of the top of stem-loop I was altered substantially. The RNase T1 cleavage at G-327 and G-328 was lost with excess Ps14 (Fig. 3, lane 4; Fig. 4C). Alternatively, the enzyme cleaved G-321 strongly. In addition, U-318, A-319, A-322 were cleaved by RNase PhyM, and A-323 and C-320 were cleaved by RNase CL3. However, the cleavage patterns of the stem I region were not changed. These results indicate that Ps14 and Rep RNA293 form a complex by base pairing between their complementary sequences. This complex is very similar in structure to RNA206, a derivative of Rep RNA293 truncated 3'-terminally at the same residue as Ps14 (14). RNA206 was unable to bind Inc RNA (19), consistent with our finding that the preincubation of Rep RNA293 with Ps14 blocked its binding to full-length Inc RNA.
The results shown in Fig. 3, lanes 2-4, indicate that the
loss of RNase T1 cleavage at G-327 and G-328 (located at the top of
structure I) is a good indicator of formation of the late complex with
Ps14. In order to measure the equilibrium dissociation
constant (Kd) for this Ps14-Rep
RNA293 complex, we incubated a fixed amount (40 nM) of 32P-Rep RNA293 with 25-400
nM Ps14 and conducted partial cleavage with
RNase T1. The amount of cleavage products at G-327 and G-328 was
quantitated relative to that of cleavage product at G-360 as an
internal standard (see Fig. 3). As shown in Fig.
5, the cleavage signal at G-327 and G-328
was reduced to 50% when [Ps14] is 80 nM,
giving the Kd value of 60 nM. Thus, the
formation of the extended pseudoknot is probably a slow process. These
results support the idea that the pseudoknot induced in vivo
by repY translation is unfolded by the inhibitory secondary
structure (III) before it reaches the more extended pseudoknot, as
illustrated in Fig. 1, steps b-d.
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Secondary Structure Analyses of the Late Complex between RepZ
mRNA and Inc RNA54--
In the presence of Inc
RNA54, the structure of stem-loop I was altered more
dramatically (Fig. 4B). The RNase T1 cleavage at G-327 and
G-328 was significantly reduced by increasing the amount of Inc
RNA54 (Fig. 3, lanes 5 and 6).
However, the RNase T1 cleavage at these residues remained even with
excesses Inc RNA54, whereas it was lost completely with
excess Ps14 (Fig. 3, lanes 4 and 6).
Consistent with this observation, only RNase PhyM weakly cleaved at
U-326 adjacent to guanine residues, among other single-strand specific
RNases (Fig. 3, lane 21). The RNase V1 cleavage at
C-312~U-314 completely disappeared and instead shifted to loop bases
at G-321 and A-322 (Fig. 3, lane 11). These results suggest
that Inc RNA54 and Rep RNA293 form a novel
X-shaped complex with two intra- and intermolecular helices, as shown
in Fig. 4B. It is conceivable that the initial base pairing
propagates in both directions until it meets with the stems of Inc RNA
and the target and, thereafter, the long intermolecular helix was
separated at the center of the loops. This separation would allow the
intramolecular stems to stand side by side, being stimulated by a
stacking trend between each of inter- and intramolecular helices.
Furthermore, the loss of RNase V1 sensitivity at C-312~U-314 strongly
suggests that the region of extended base pairing covers the 21-base
Inc loop region plus a few bases each from the upper stem and includes the bases different from IncB pMU720 plasmid (Fig. 4B).
Because the bases in the 5' single-stranded leader of the antisense
RNAs are identical between IncI and IncB plasmids, we propose that the stability of the complex as depicted in Fig. 4B is
responsible for determining different incompatibility between IncI
and IncB plasmids (see under "Discussion").
Next we attempted to estimate the Kd value for this late Inc RNA54 complex by plotting the relative intensity of the RNase T1 cleavage products at G-327 and G-328 against [Inc RNA54]. Fig. 5 shows that Kd for the Inc RNA54 complex is much smaller than that for the Ps14 complex and is close to the detection limit for this experiment. Nevertheless, we could tentatively estimate the Kd value of ~3 nM. Although determining the accurate Kd value awaits further experiments, the results in Fig. 5 suggest that the late Inc RNA54 complex is more stable than the transient initial interaction (C*) and corresponds to the C** complex. Thus, the whole structural data support our model that the initial base-paring between the stem-loops of Inc RNA54 and the target in Rep RNA293 (C*) rapidly transforms into a more stable, irreversible complex (C**), leading to block full-length Inc RNA binding, as observed in Fig. 2A.
Why Is the Late Inc RNA54 Complex Unstable?--
The
intermolecular base pairing observed in the late Inc RNA54
complex was quite extensive as shown in Fig. 4B.
Nevertheless, it was not stable in denaturing gels, probably because
the interacting loops are closed by stable stems: annealing of two
strands of RNA generates torsion as they form a helical structure.
Fixing the ends of these strands by a stable stem would remove the way to release such torsion, thereby providing a stress into the structure of the resulting complex. To test this idea briefly, we prepared Inc21, a 21-base RNA corresponding to the loop of Inc RNA,
and allowed it to bind to Rep RNA293. Inc21
does not form any secondary structure (data not shown) and therefore
would wind up to loop I without storing a stress. As expected, this RNA
rapidly formed a stable complex, persistent in denaturing gels (Fig.
6). Because the Inc RNA54-Rep
RNA293 complex was separated in the same denaturing gel
conditions (16), we conclude that having a loop closed by a stable stem
indeed destabilized the loop-loop complex. The stable binding of
Inc21 to RepZ mRNA also supports our previous proposal that there is little requirement in the structure of Inc RNA for its
efficient binding to RepZ mRNA (16, 19).
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Molecular Basis for Differential Control of repY and repZ by Inc RNA-- The plasmid ColIb-P9 Inc RNA controls translation of repY and repZ with different mechanisms (12). By using Inc RNA54 lacking the 5' leader of Inc RNA and Ps14, a part of RepZ mRNA complementary to loop I, we previously showed that the initial loop-loop base pairing between Inc RNA and the target in the repZ mRNA leader can compete with formation of the pseudoknot required for repZ translation (16). In this report, we show that Inc RNA54 not only reduces the initial rate of the Inc RNA-RepZ mRNA hybridization by its transient base pairing to loop I, but also stops the reaction by its subsequent irreversible binding to the loop (Fig. 2). Intermolecular base pairing found in the latter irreversible complex was quite extensive, spanning probably > 20 base pairs and forms two helices separated at the site of the initial base pairing (Figs. 3 and 4). The equilibrium dissociation constant for this complex was tentatively estimated to be ~3 nM (Fig. 5), a value smaller than that for the transient initial interaction, 6 or 10 nM (16). Nevertheless, the late complex was unstable in denaturing gels, probably because of a structural stress stored during the complex formation (Fig. 6). Based on these results, we propose that the stepwise model for antisense RNA binding proposed for the ColE1 system (class H) (Ref. 18; see the Introduction) also applies to a class L antisense RNA, but with completely different molecular mechanisms. We believe that the late Inc RNA54 complex characterized here as C** corresponds to an intermediate complex formed between IncB pMU720 RNA T (a 5'-truncated derivative) and its target that was analyzed by native gel electrophoresis (Ref. 23; see below).
In view of differential control of repY and repZ, several important functions could be assigned to the late intermediate step, as depicted in Fig. 1, step g. First, it is conceivable that the observed irreversible base pairing would trap the initial transient interaction in a bound state, thereby driving the binding reaction. Second, for the control of repZ, the inhibition of the pseudoknot formation should be established at this stage, because the bases critical for the pseudoknot formation (G-327~G-330) are still masked (Fig. 4B). Furthermore, the presumed side-by-side configuration of the complex would bring the ends of the stems closer, thereby allowing the Inc 5' leader (absent in Inc RNA54) to base pair efficiently with a region distal to the repY RBS (Fig. 1, steps g and h).
The antisense RNA I of IncB pMU720 replicon is identical to the Inc RNA
of IncI ColIb-P9 except at the bases indicated in Fig.
4B. Therefore, the partitioning of these plasmids into
different incompatibility groups (22) should be explained at the level of the C** complex formation as analyzed in this study. It is also
noteworthy that the mutant IncI Inc RNA with a loop mutation (G329'A
in ColIb-P9) abolishing the C* formation (16) showed no detectable
incompatibility (or interaction) against both wild-type IncI and
IncB plasmids (22). Thus, the mutation in the C* region has more
dramatic effect on plasmid incompatibility than that located outside
affecting only C** formation, consistent with our previous results
(19).
Complex C** Formed by Other Class L Antisense RNAs-- Although the presence of C* has not yet been demonstrated for IncFII plasmids, an intermediate complex, called the "extended kissing complex," was extensively analyzed with the CopA antisense RNA encoded by the R1 plasmid of IncFII group (24-27). This complex, formed between a derivative of CopA lacking its 5' leader and the CopA target, was often characterized by native gel electrophoresis and gave the Kd of 7.4 nM (26). In light of our study, we believe that this kissing complex corresponds to C** for the following reasons. First, 5'-truncated CopA had been incubated with the target mRNA for more than 5 min to form this complex (24-27); this time is long enough to allow progression to C**, based on our results (Fig. 2). Second, reduced but remaining RNase T2 sensitivity at a loop G residue found in the 5'-truncated CopA in complex with the target (24) compares very well with the weak RNase T1 sensitivity at G-327 and G-328 found in the structure I of RepZ mRNA in complex with Inc RNA54 (Fig. 3). Third, a measurement by native gel electrophoresis may underestimate the Kd value, because the complex can dissociate in the gel. In fact, Hjalt and Wagner (26) realized this problem and measured Kd differently by incubating the target RNA with various concentrations of the 5'-truncated CopA and measuring the amount of free form of the former that can bind intact CopA RNA: Kd thus obtained was 1.6 nM consistent with our preliminary estimation for C** (~3 nM; Fig. 5). These values are significantly higher than the Kd obtained for C** in ColE1, 0.1 nM (18). This fact agrees with the idea that C** in ColIb-P9 (or R1) is destabilized by a structural stress imposed by the propagation of a single initial loop-loop interaction (Fig. 6). C** in ColE1 should be free of such a stress, because it is composed of multiple loop-loop interactions involving a smaller number of base pairs (18).
Finally, the secondary structure of the extended kissing complex was analyzed with Pb(II)-induced cleavage experiments (27). Malgren et al. (27) appear to assume that the extended intermolecular base pairing occurs only at the 3' side of the target loop, even though Pb(II) sensitivity was also reduced at the 5' side. Using four different RNases that have potential to cleave all four kinds of nucleotides plus a double-strand specific RNase V1, we unambiguously showed that both sides of the partially base paired target loop were protected by Inc RNA54 (Fig. 4B). In sharp contrast, the 5' side of the target loop in complex with Ps14 showed dramatically increased sensitivity to RNases T1 and PhyM (Fig. 4C). Another new aspect concerns the weak single-stand specific RNase sensitivity at U-326~G-328 (Fig. 3). These results raised the interesting possibility that the intermolecular helix is separated at the top of structure I, resulting in a stacking trend between each separated helix and the stem of Inc RNA or structure I (Fig. 4B). We already discussed the biological significance for this unique configuration as above.
Evolution of Control of Plasmid Replication by Class L
Antisense RNAs--
The whole pathway of Inc RNA binding as shown in
Fig. 1 appears to fit best for its differential control of
repY and repZ expression. Antisense RNAs encoded
by IncFII plasmids also follow a similar binding pathway (27).
Nevertheless, the translation of rep genes encoded by these
plasmids does not require formation of a pseudoknot (9, 28), unlike the
case for IncI (13) and IncB (15) plasmids. Instead, IncFII plasmids
additionally encode a transcriptional repressor for the rep
mRNA, located upstream of the repressible promoter (3) (see Fig.
7). How did these differences arise
during the course of evolution?
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We believe that the difference between these related but distinct types
of plasmids arose at least partly from difference in RNA secondary
structure that blocks the RBS for respective rep genes, as
depicted in Fig. 7B. In the case of IncI/IncB-type, the
secondary structure that blocks the rep RBS is very tight. Therefore the translation of rep is possible only when the
pseudoknot induced during the RLP translation termination transiently
keeps the rep RBS accessible to the ribosome (6, 14, 16). On the other hand, the inhibitory secondary structure found in IncFII plasmid replicons is less tight around the rep RBS. Thus,
the refolding of the inhibitory secondary structure, unfolded during the RLP translation termination, would be slow enough to allow the
ribosome to bind the rep RBS. However, this kind of
translational coupling could enable leaky, unregulated translation
initiation of rep, as demonstrated for a mutant ColIb-P9
replicon lacking the pseudoknot and carrying less tight structure III
(rep inhibitory stem-loop) (6). It is therefore conceivable
that the ancestor of IncFII-type plasmids acquired an additional
control system at the transcriptional level, in order to complement
this regulatory defect.
It would be difficult to solve the evolutionary relationship between
these regulatory systems. Nevertheless, we favor the model that the
pseudoknot-mediated control in IncI/IncB plasmids is more ancient
and was lost in the ancestor of IncFII plasmids during recombination
between plasmids. In support of this scenario, the "mosaic"
structure of large, self-transmissible plasmids is not uncommon (20),
and there are even some cases of naturally occurring hybrid replicons
carrying the control region of IncI/IncB-type and the rep
gene of IncFII-type, or vice versa (9). Evolution of plasmids by
recombination may have been stimulated by horizontal transfer of
plasmids between a variety of bacteria encoding different restriction-modification systems (29). On the other hand, certain types
of plasmids that are transferred only between limited species of
enterobacteria and not exposed to harsh natural environment might
retain the intact plasmid genome and hence the "molecular fossil"
of replication control system. The ongoing genome-wide sequence
analyses for different groups of self-transmissible plasmids may answer
whether ColIb-P9, originally isolated from Shigella sonnei,
is one such plasmid.
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ACKNOWLEDGEMENT |
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We are indebted to Shigeyuki Yokoyama for inspiring discussion and the experiments conducted by K. A. at the University of Tokyo. We thank Alan Hinnebucsh for comments on the manuscript, Tatsuya Niimi for sharing materials used for RNase cleavage experiments, and members of the Yokoyama and Mizobuchi laboratories for helpful discussion.
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FOOTNOTES |
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* This research was supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture (Monbu-sho) 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 the 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@aghmac1. nichd.nih.gov.
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ABBREVIATIONS |
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The abbreviations used are: Inc, incompatibility; RLP, rep leader peptide; RBS, ribosome-binding site.
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REFERENCES |
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ABSTRACT
INTRODUCTION
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REFERENCES
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| 1. | Eguchi, Y., Itoh, T., and Tomizawa, J. (1991) Annu. Rev. Biochem. 60, 631-652[CrossRef][Medline] [Order article via Infotrieve] |
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| 3. | Nordström, K. (1990) Cell 63, 1121-1124[CrossRef][Medline] [Order article via Infotrieve] |
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Novick, R. P.
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