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 sequence 5'-rUUGGCG-3' is conserved within
the loop regions of antisense RNAs or their targets involved in
replication of various prokaryotic plasmids. In IncI plasmid
ColIb-P9, the partially base paired 21-nucleotide loop of a stem-loop
called structure I within RepZ mRNA contains this hexanucleotide
sequence, and comprises the target site for the antisense Inc RNA. In
this report, we find that the base pairing interaction at the
5'-rGGC-3' sequence in the hexanucleotide motif is important for
interaction between Inc RNA and structure I. In addition, the 21-base
loop domain of structure I is folded tighter than predicted, with the hexanucleotide sequence at the top. The second U residue in the sequence is favored for Inc RNA binding in a base-specific manner. On
the other hand, the upper domain of the Inc RNA stem-loop is loosely
structured, and maintaining the loop sequence single-stranded is
important for the intermolecular interaction. Based on these results,
we propose that a structural feature in the loop I domain, conferred
probably by the conserved 5'-rUUGGCG-3' sequence, favors binding to a
complementary, single-stranded RNA. This model also explains how the
RepZ mRNA pseudoknot, described in the accompanying paper (Asano,
K., and Mizobuchi, K. (1998) J. Biol. Chem. 273, 11815-11825) is formed specifically with structure I. A possible conformation adopted by the 5'-rUUGGCG-3' loop sequence is
discussed.
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INTRODUCTION |
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In prokaryotes, small antisense RNAs, encoded by the antisense strand of the transcribed leader region, negatively control the expression of many genes at the transcriptional or post-transcriptional level, or the formation of primers for plasmid DNA replication (for review, see Refs. 1 and 2). In ColE1 and related plasmids, the antisense RNA I and its target on the pre-primer RNA II each comprise three small stem-loop structures with 6 to 7 base loops complementary to each other, and a transient interaction between these loops is rate-limiting for interaction between RNAs I and II (1).
The replication of low-copy number IncFII, IncB, and IncI group
plasmids is also negatively controlled by antisense RNAs, which in
these cases bind to the complementary regions in the transcribed
leader sequences of the genes encoding replication initiator proteins
(1, 2). Antisense and target RNAs of these plasmids essentially carry
only one stem-loop structure. The sequence 5'-rUUGGCG-3' is conserved
within the loop regions of the target RNA (3). Besides these plasmids,
the same hexanucleotide sequence is found in the loop of the ColE2
antisense RNA I which controls the replication of this high-copy number
plasmid (4), and a similar 5'-rUUCGCG-3' is located in the loop of the
antisense RNA-OUT, which controls translation of the Tn10 transposase
(5). In all of these cases so far analyzed, base pairing interaction between the two complementary RNA molecules at the G/C-rich segment of
3 or 4 bases within the conserved hexanucleotide sequence is rate-limiting for the intermolecular interaction (4-6).
In IncI plasmid ColIb-P9, it is predicted that both the antisense
Inc RNA and its target, structure I, within the RepZ mRNA leader
region are folded into a single large stem-loop with the size of 51 bases (7, 8). Both of these stem-loops are divided into two domains:
the bottom contiguous stem of 15 base pairs, and the large partially
base paired loop of 21 bases (Fig. 1). Structure I of RepZ mRNA
contains the conserved 5'-rUUGGCG-3' sequence at the top of the upper
loop domain at positions 325-330 (boxed by dotted
line in Fig. 1). Four single base substitutions in each nucleotide
of the 5'-rGCCA-3' sequence of Inc RNA (positions 329'1 to 326', shown by
arrowheads in Fig. 1) reduce the ability to repress
repZ translation, suggesting that base pairing interaction at the conserved sequence is again important for interaction between the antisense and the target RNAs in the ColIb-P9 system (9). In
addition, it is at this 5'-rUUGGCG-3' site that the RepZ mRNA pseudoknot required for repZ translation is formed with the
5'-rCGCC-3' sequence separated by 107 bases at positions 437-440
(Refs. 9-11; also see Fig. 1). These observations have strengthened
the importance of this conserved loop sequence for RNA-RNA base pairing
interactions, and suggested to us that the positive and negative
regulatory elements for repZ gene expression (the RepZ
mRNA pseudoknot and Inc RNA) function in a competitive manner.
In this report, we extended our analyses into interaction between Inc RNA and the structure I of RepZ mRNA. Our studies not only suggest the importance of the conserved 5'-rUUGGCG-3' sequence in structure I for stimulating binding of a complementary, single-stranded RNA, but also explain how the RepZ mRNA pseudoknot, described in the accompanying paper (11), is formed specifically with structure I. Evidence is also presented suggesting that the formation of RepZ mRNA pseudoknot and the binding of Inc RNA are mutually exclusive events in vitro and function competitively in vivo.
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EXPERIMENTAL PROCEDURES |
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Bacteria, Phages, and Plasmids--
Escherichia coli
K12 strains used in this study were W3110,
W3110ind
, MV1184 (12), NM522 (13), BW313
(14), BMH71-18mutS (15), and MC1061 (Lac
) (16). Plasmids
used in this study are listed in Table I. pKA340-W3 and its mutant derivatives were translational repZ-lacZ fusions cloned in a single-copy vector, mini-F, and employed as the recipient plasmid for Inc RNA in vivo. pKA18 and its
mutant derivatives were used as the donor of Inc RNA. pKA10 and its
mutant derivatives were used for in vitro synthesis of Inc
RNA and the leader region of RepZ mRNA.
:ColIb-P9 hybrid phage
CH10W was constructed by subcloning the 3.0-kilobase
EcoRI fragment of pAK10 into the EcoRI site of
VIII (20).
W1 is a spontaneous copy-up mutant of ColIb-P9
isolated from
CH10W (see below).
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Isolation of Copy-up Mutants Using :ColIb-P9 Hybrid
System--
The procedure for isolation of copy-up mutants using
:ColIb-P9 hybrid was described (7). As the original
:ColIb-P9
hybrid,
CH10 (7), was found to carry a moderate copy-up mutation, C334A, we re-screened copy-up mutants using the same scheme with the
wild-type
:ColIb-P9 hybrid phage,
CH10W. Clear-plaque forming mutants of
CH10W plated on W3110
ind
were
isolated at a frequency of 6 × 10
7, and purified.
Among 12 independent mutants, six carried a base substitution in the
inc gene promoter. We located two A400C (inc1) and two A400G mutations in the
35 region, and one T376C and one A372G
mutations in the
10 region. Five other mutations represented as
W1
changed T-325 in the inc loop to C (T325C), and still
another had an insertion of A between positions 398 and 399. This
mutation fuses repY to repZ in-frame, and also
affected the inc gene promoter. We used
W1 carrying a new
inc loop mutation for further study.
Determination of Plasmid Copy Numbers by Gel Electrophoresis-- MC1061 cells carrying each 4.3-kilobase mini-ColIb-P9 plasmid were grown exponentially to A600 of 0.40 at 37 °C in LB medium supplemented with kanamycin (40 µg/ml). Plasmid DNA was isolated according to Birnboim and Doly (21). After the cells were suspended in solution II, 1.2 µg of pKA140-I2 with a size of 11 kilobases (9) was added to each sample as an external standard to monitor the efficiency of DNA extraction. A portion of DNA samples was linearized by digestion with BstP1 which cleaves both mini ColIb-P9 and pKA140-I2 uniquely, and electrophoresed on a 0.7% agarose gel containing ethidium bromide. The gel was photographed under UV light. The negative was scanned by Shimadzu CS930 spectrophotometer.
Measurement of -Galactosidase Activity--
MC1061 cells
carrying the wild-type or mutant pKA340-W3 and pACYC184, pKA18, or its
mutant derivative were grown in the presence of kanamycin (40 µg/ml)
and chloramphenicol (50 µg/ml) and assayed for
-galactosidase
activity as described (9). The specific activity of the enzyme was
expressed as Miller units (22). The values reported here are the
results of at least three independent experiments, and the standard
deviations were within 15% of each value.
Preparation of RNA--
Mutant derivatives of the RepZ mRNA
leader region were synthesized using T7 RNA polymerase essentially as
described (11). Unlabeled RNA293 was prepared from the same
reaction mixture except that 2 mM GTP replaced 0.5 mM GTP, and that 0.222 MBq of [5,6-3H]UTP
(1000 GBq/mmol) was used instead of [-32P]GTP for
quantitation. 5'-32P-RNA120 was prepared with
EcoO109I-digested pKA10 as the template. Mutant derivatives
of these RNAs were synthesized using respective mutant derivatives of
pKA10.
Measurement of Binding Rate Constants in Inc RNA-RepZ mRNA
Leader Region Hybridization--
Binding rate constants between Inc
RNA and RepZ mRNAs were determined in the standard binding buffer
as described (24) with slight modification. 2.5 × 1010 to 1 × 10
9 M of
uniformly 32P-labeled Inc RNA was incubated with 2.5 × 10
9-10
7 M unlabeled
RNA293 in the binding buffer at 37 °C. Molar
concentrations of RNA293 were always more than 10 times
higher than those of 32P-Inc RNA. Aliquots were withdrawn
at various times and mixed with an equal volume of an ice-cold solution
(95% formamide, 0.1% xylane cyanol, 0.1% bromphenol blue, 17 mM EDTA). Reactions were analyzed by electrophoresis on a
8% polyacrylamide gel containing 8.3 M urea in TBE buffer
(23). Autoradiography was at
70 °C with a Kodak X-AR5
film.
RNase Digestion-- The 5' 32P-labeled Inc RNAs and RNAs120 (0.06 µg/reaction) were subjected to secondary structure analysis by RNase T1, RNase derived from Bacillus cereus, RNase Bc, or RNase V1 as described (11).
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RESULTS |
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Mutations Affecting the Negative and Positive Control of repZ
Expression--
Mutations changing each base of the 5'-UUGGCG-3'
sequence (positions 325-330), as shown in Fig.
1, were isolated spontaneously or
introduced by site-directed mutagenesis (see "Experimental Procedures" and Ref. 11). We employed mutations C334A and G331A since
they are expected to disrupt the internal base pairing within the loop
domains of structure I and Inc RNA. C334A was found fortuitously during
the construction of a mini-ColIb-P9 plasmid, pCH10. C334A and C329T
were moderate copy-up mutations increasing copy number 9- and 5-fold
compared with wild-type,
respectively.2 T325C was
found in a clear plaque-forming mutant, designated W1, derived from
the
:ColIb-P9 hybrid phage,
CH10W (see "Experimental Procedures"). Copy number of the ColIb-P9 replicon excised from
W1
was increased 36-fold compared with wild-type (data not shown). repZ expression was increased 75-fold by T325C when measured
by
-galactosidase activity with a repZ-lacZ translational
fusion carried by pKA140-W1 in high copy (data not shown). In contrast to these three copy-up mutations, rep2006 (G327A), G328A,
and rep2041 (G330A) impair replication due to a failure to
form the pseudoknot required for repZ expression (9, 11).
G331A also reduced the level of repZ expression
significantly in the absence of Inc RNA (9, 11).
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Effects of inc Loop Mutations on Negative Control of repZ Gene Expression-- To analyze effects of inc loop mutations on negative control of repZ expression without affecting positive control, the repZ-lacZ reporter plasmids and Inc RNA donor plasmids were constructed as described under "Experimental Procedures." The reporter plasmids, pKA340-W3 and its mutant derivatives, carry the inc1 mutation to eliminate production of endogenous Inc RNA. In addition, formation of the pseudoknot required for repZ translation was maintained in the reporters by introducing base changes in the 5'-TCCGCCA-3' sequence at positions 435-441 that compensate for the inc target mutations in structure I, e.g. sup2006-10 (C440T) for rep2006 (G327A) (9). As shown in Table II, the level of repZ expression from the reporter plasmids with different Inc RNA target sites (pKA340-W3 derivatives) are between 706 and 2177 units due to these secondary mutations in the absence of inc encoded on the donor plasmid (vector alone, pACYC184) (Table II, the first line in each mutant subset). This level of expression is comparable to 1978 units from pKA340-W3 encoding wild-type structure I (Table II, line 1, column 5).
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Characterization of inc Loop Mutations by Means of the Inc
RNA-RepZ mRNA Hybridization Method--
To further analyze the
intermolecular interaction between Inc RNA and RepZ mRNA, we
synthesized in vitro 32P-Inc RNA and
RNA293, a part of the RepZ mRNA leader region, from wild-type and mutant derivatives. The mutations used are identical to
those employed in Table II (see Fig. 1). To measure the degree of the
intermolecular interaction, the wild-type 32P-Inc RNA
(2.5 × 1010 M) was incubated with
excess amounts (2.5 × 10
9-1 × 10
7 M) of unlabeled wild-type
RNA293. A portion of the mixture was withdrawn at various
times and analyzed electrophoretically on a denaturing polyacrylamide
gel. The pattern of hybridization of 32P-Inc RNA in the
presence of 1 × 10
8 M
RNA293 is shown in Fig.
2A. When the relative amount
of unhybridized Inc RNA was plotted as the function of incubation time,
the rate of RNA-RNA hybrid formation followed the pseudo first-order
kinetics (Fig. 2B). The apparent binding rate k',
defined as ln 2/t1/2, where t1/2
is time in seconds required to bind one-half of the Inc RNA, was
proportional to the molar concentration of RNA293 (data not shown). Therefore, we obtained the binding rate constant k,
defined as k'/[RNA293], to be 6.8 × 105/M/s. This value was comparable to those
obtained for other antisense RNA systems such as ColE1 RNA I, R1 CopA,
Tn10 RNA-OUT, ColE2 RNA I, or IncB pMU720 RNA I (1, 6).
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The Secondary Structure of Wild-type and Mutant RepZ mRNAs-- We analyzed the secondary structure of stem-loop structure I, the target site of Inc RNA, in the accompanying paper (11). When 5'-labeled RepZ mRNA leader region synthesized in vitro was probed with RNase T1 that specifically cleaves after guanine residues in single-stranded regions, we observed major cleavages only at G-327 and G-328, suggesting that the entire region, including most of the upper loop domain of 21 bases, is folded so that the 5'-rGGC(G)-3' sequence, critical for base pairing with Inc RNA, is located in the loop (Ref. 11; also see Fig. 3, Wild-type). However, minor cleavages by RNase Bc (specific for pyrimidine residues in single-stranded regions) at C-324 and U-325 suggest that the base pairing in the upper loop domain is not very tight (Fig. 3). A preliminary nuclear magnetic resonance study supports the presence of a G:U wobble base pairing in structure I.3
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Structure Surrounding the 5'-rUUGGCG-3' Motif in RepZ mRNA Is Important for Inc RNA Binding-- All the type II mutations changed the RNase cleavage patterns in structure I (Fig. 3A). The base substitution of G331A induced a new RNase T1 cleavage at G-330 compared with wild-type (Fig. 3A), suggesting unfolded conformation in the upper loop domain of structure I (Fig. 3B). Mutation C334A yielded additional RNase T1 cleavages at G-321, G-331, and G-335, compared with wild-type (Fig. 3A). This observation implies that the whole loop domain of structure I was unfolded although the G-330 residue was still protected from RNase T1 probably by weak base pairing with U-325 (Fig. 3B). Moreover, mutation T326C created new RNase T1 sensitivity at G-321 and G-331, again suggesting destabilization of the upper loop domain (Fig. 3, A and B). Note that the extent of disruption of the internal base pairing between 5'-rCU-3' (positions 324 and 325) and 5'-rGG-3' (positions 330 and 331) in the upper loop of structure I seemed to correlate with the degree of reduction in binding wild-type Inc RNA (Table III, lines 6, 24, and 27), suggesting the importance of the internal base pairing for Inc RNA binding. Consistent with this idea, stabilizing this base pairing by changing U-325 to C (T325C in Fig. 3) did not affect interaction with wild-type Inc RNA significantly (Table III, line 3).
Of note is the finding that mutation T326G appeared to confer even tighter secondary structure in the upper loop domain of structure I unlike the other type IIa mutation, G331A (Fig. 3B), as suggested by decreased RNase Bc cleavages at C-324 and U-325 (Fig. 3A). This result indicates that the altered U-326 is important for binding Inc RNA in a base-specific manner. Here we should note that U-326 corresponds to the second base in the conserved 5'-rUUGGCG-3' sequence and that the internal base pairing as mentioned above gives rise to the loop of U-326 plus the 5'-rGGC-3' sequence important for base pairing with Inc RNA. How the T326G mutation caused defect in Inc RNA binding will be discussed later in relation to the possible loop conformation adopted by this unique RNA motif.The Secondary Structures of Wild-type and Mutant Inc RNAs--
We
then analyzed the secondary structures for wild-type and mutant Inc
RNAs using 5'-labeled 32P-Inc RNA. Inc RNA was prepared
in vitro using E. coli RNA polymerase. When we
used [-32P]GTP or [
-32P]ATP for
labeling Inc RNA during the in vitro transcription reaction, it was labeled only with [
-32P]GTP (data not shown),
indicating that the transcription of Inc RNA starts at a G residue
in vitro. The labeled Inc RNA was purified by gel
electrophoresis and the secondary structure was analyzed using RNase
T1, RNase Bc, and RNase V1. The cleavage patterns of wild-type Inc RNA
are shown in Fig. 4A. RNase T1 cleaved
strongly at G-329', moderately at G-324' and G-358', and weakly at
G-334' and G-320' (lane 2). The comparison of this RNase T1
cleavage pattern with the alkaline cleavage ladder of Inc RNA
(lane 1) localized the transcriptional start of Inc RNA to
G-365' and revealed its size to be 65 to 67 bases. The 5'-end of Inc
RNA isolated in vivo was two bases shorter, starting at
A-363' (8), whereas its 3'-end was seven bases longer, ending mainly at
T-292'.4 We do not know the
exact reason for this discrepancy, but these could be due to
modification in vivo, such as endonucleolytic cleavage and
polyadenylation, as observed in ColE1 RNA I (25).
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Competition between the Pseudoknot Formation and Binding of Inc RNA-- Structure I is not only the target site for Inc RNA binding, but also the site of formation of the pseudoknot with the 5'-rCGCC-3' sequence at position 437-440, which is required for repZ translation (9-11). Thus, we asked if binding of Inc RNA and formation of the pseudoknot compete with each other. For this purpose, RepZ mRNA derivatives that formed the pseudoknot at different degrees depending on the presence of inhibitory secondary structures (11) were hybridized with wild-type Inc RNA in vitro. RNA293 chosen as starting material does not form a pseudoknot, since the 5'-rCGCC-3' sequence at position 437-440 is masked by the stable stem-loop structure III (11). The presence in each RNA species of pseudoknot or the secondary structure preventing it from forming is indicated in Table IV, columns 2-4. As shown in Table IV, column 5, RNA293 bound Inc RNA rapidly regardless of the presence of mutation rep2044 disrupting the pseudoknot formation. RNA206, which formed the pseudoknot predominantly, did not bind Inc RNA. The introduction of rep2044 recovered the rate constants of RNA206 to the level of RNA293 (in rep2044 RNA206). When the pseudoknot is formed partially due to the presence of another inhibitory secondary structure IIIa (A25 RNA293), or due to removing a part of the complementary sequence (A28 RNA290), RepZ mRNA leader bound Inc RNA at reduced rates. These reduced rates were recovered again to the wild-type level when rep2044 was introduced in the mutant RNAs (A32 RNA293 or A34 RNA290 as rep2044 derivatives of A25 RNA293 or A28 RNA290, respectively). These results indicate that the formation of RepZ mRNA pseudoknot physically interferes with the binding of Inc RNA, probably by masking the 5'-rGGCG-3' sequence.
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DISCUSSION |
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In this report, we investigated the structural basis for Inc RNA-RepZ mRNA interaction by examining the effects of inc loop mutations on repZ expression (Table II), the rate constant of in vitro binding (Table III) and secondary structure of both Inc RNA and its target on RepZ mRNA, the stem-loop structure I (Figs. 3-5), and showed that base pairing at the 5'-rGGC-3' sequence in the conserved 5'-rUUGGCG-3' sequence at the top of structure I is rate-limiting for the intermolecular interaction. Similar base paring interaction between the three nucleotide sequences was proposed previously in a closely related IncB plasmid replicon (6). We believe that the critical base pairing interaction occurs initially to give a high specificity to the Inc RNA-RepZ mRNA interaction, as demonstrated for the antisense control systems in ColE1 and IncFII R1 replicons (1, 2). By using a part of Inc RNA that does not form a persistent hybrid with RepZ mRNA, we demonstrated that such an initial contact indeed exists in the ColIb-P9 control system.2
In addition, secondary structure analyses of Inc RNA and RepZ mRNA in both wild-type and mutants (Figs. 3-5) suggested to us strongly that the partially base paired loop conformation within RepZ mRNA structure I was important for the interaction. The second U residue in the conserved 5'-rUUGGCG-3' sequence, located 5' to the 5'-rGGC-3' sequence, was also important in a base-specific manner. By contrast, the 21-base long loop region of Inc RNA was basically unstructured. The unstructured nature of Inc RNA loop probably serves to keep the sequence complementary to the conserved hexanucleotide sequence as single-stranded. Thus, the conserved sequence, as a novel RNA motif, appears to adopt a specific RNA conformation and stimulate its binding to a single-stranded, complementary RNA. We also presented the evidence suggesting that the formation of RepZ mRNA pseudoknot and the binding of Inc RNA are mutually exclusive events in vitro (Table IV) and function competitively in vivo (Table V). Based on these findings, it could be proposed that the ability of the specific sequence in the loop of structure I to bind to either Inc RNA or a downstream sequence within RepZ mRNA is important for determining the level of repZ gene expression.
What kind of physical structural properties are given to structure I by
this unique 5'-rUUGGCG-3' motif? Since the bases in the 21-base
long loop of structure I located 3' to the second U residue in the
hexanucleotide sequence are rich in purines (8 G or As/11 bases), they
may stack together on the stem part of structure I in an A-form helix.
In this model, the second U residue (U-326) provides the -turn of
the phosphate backbone and stabilizes the base stacking (Fig.
6B). This would be analogous
to the anticodon loop of tRNAs with a conserved U residue located 5' to
the anticodon triplet (Refs. 26-28; see Fig. 6A), and also
the human immunodeficiency virus TAR RNA hairpin with a loop of
5'-rCUGGGA-3' (29). Weak wobble base pairing between the first and
sixth residues in the hexanucleotide sequence and a few more base pairs
within the upper loop of structure I likely contribute to maintaining
this proposed structure.3 Thus, the single-stranded
5'-rGGC-3' sequence located at the 5'-end of the putative helix would
be available to base pair with its counterpart sequence in Inc RNA, as
illustrated in Fig. 7B, steps a and b. This model can explain why
replacing U-326 with a G residue impairs binding between Inc RNA and
T326G RepZ mRNA (Table II, line 13, and Table III,
line 9), as placing a bulky purine residue at U-326 is
expected to disturb the helical nature of structure I. On the other
hand, the 5'-rCGCCAA-3' sequence within Inc RNA, complementary to the
hexanucleotide motif, lacks both of a U residue providing the
-turn
and wobble base pairing between the first and sixth residues, leading
to the unstructured nature of the Inc RNA loop. Thus, replacing A-326',
equivalent of U-326 in Inc RNA, to a C residue has little effect on Inc
RNA pairing with structure I (Table II, line 14, and Table
III, line 10).
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Internal loops or "bulged-out" nucleotides are commonly observed in
the upper loop domains of the various antisense and target RNAs
carrying the conserved 5'-rUUGGCG-3' sequence (Fig. 7A). In
CopA and its target RNAs of plasmid R1, the destabilization of the loop
domains conferred by this feature is important for allowing the initial
base pairing interaction to propagate by a few more base pairs, thereby
stabilizing transiently the initial recognition reaction (Ref. 31, also
see Fig. 7B, steps b and c). It is proposed that
pairing between the antisense RNA I and its target in IncB pMU720
replicon, closely related to IncI ColIb-P9, proceeds in analogous
steps (6). In addition to these bulged-out nucleotides, the importance
of structural differences between the partially base paired loop
domains of antisense and target RNAs, as in this study, have been
suggested by the mutational analyses of the ColE2 and IncB plasmid
replication control systems (4, 30). We believe that these structural
differences result from the helical conformation of the 3'-side of the
loop in one RNA, given by the 5'-rUUGGCG-3' motif and base stacking,
and lack of important secondary structure in the other, as proposed
here. In this way, the partially base paired loop sequence carrying this motif confers specificity to the initial interaction with the
complementary RNA, allowing at the same time subsequent propagation of
the interaction for stabilizing the complex (Fig. 7B).
We also believe that formation of the RepZ mRNA pseudoknot involving structure I and the 5'-rCGCC-3' sequence, located 85 bases downstream at positions 437-440 and released by unfolding stem-loop structure III during the process of repY translation and termination (9-11), occurs similarly to the initial step in the binding of Inc RNA to structure I. Consistent with this idea, base pairing at the 5'-rGGCG-3' sequence, which overlaps with one critical for pairing with Inc RNA, is critical also for the pseudoknot formation (9, 11). Moreover, efficient formation of the pseudoknot requires the U-326 residue, the second base in the 5'-rUUGGCG-3' motif of structure I, in a base-specific manner (11). Indeed, we now have evidence suggesting that the step of the pseudoknot formation as required in vivo for repZ translation is equivalent in apparent thermodynamic stability to one in the initial base pairing interaction between Inc RNA and RepZ mRNA at the 5'-rGGC-3' sequence (positions 327-331) as described as steps b and c in Fig. 7B.2 It is therefore possible that Inc RNA could inhibit formation of the RepZ mRNA pseudoknot at the level of the initial base pairing interaction.
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ACKNOWLEDGEMENTS |
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We thank H. Moriwaki for excellent technical assistance, and Alan Hinnebusch and Robert Weisberg for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by a grant-in-aid from the Ministry of Education, Science, and Culture 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 Japan Society for the Promotion of Science Fellowships for Japanese Junior Scientists. To whom correspondence should be addressed. Present address: Laboratory of Eukaryotic Gene Regulation, NICHD, National Institutes of Health, Bethesda, MD 20892.
1 We added a prime to the ColIb-P9 coordinate in the complementary strand.
2 K. Asano and K. Mizobuchi, manuscript in preparation.
3 K. Asano, T. Niimi, S. Watanabe, S. Yokoyama, and K. Mizobuchi, unpublished observations. In a preliminary nuclear magnetic resonance study with a 54-base RNA encompassing structure I from positions 302 to 352, we observed imino-proton resonances corresponding to the stem part of 15 base pairs, and 4 additional broader ones which may correspond to the base pairings in the upper loop domain including one from a G:U base pair.
4 A. Kato and K. Mizobuchi, unpublished observations.
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REFERENCES |
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