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
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Translation initiation of the repZ gene encoding the replication initiator of plasmid ColIb-P9 is not only negatively regulated by the action of the antisense Inc RNA encoded in the leader region, but is also coupled to the translation and termination of a transcribed leader sequence, repY, a positive regulatory element for repZ gene expression. This translational coupling depends on base pairing between two complementary sequences, 5'-rGGCG-3' and 5'-rCGCC-3', which are located upstream of and in the middle of repY, respectively, and have the potential to form a pseudoknot with the stem-loop structure I. Another stem-loop called structure III near the 3'-end of repY sequesters both the 5'-rCGCC-3' sequence and the repZ ribosome-binding site. Here we show that the RepZ mRNA leader sequence synthesized in vitro indeed contains several stem-loop structures including structures I and III, but not the pseudoknot. However, disruption of structure III, without changing the repZ ribosome-binding site, by means of base substitution and deletion induces base pairing between the two short complementary sequences distantly separated, resulting in the formation of a pseudoknot. When the pseudoknot is allowed to form in vivo due to the same mutations, a maximum level of repZ expression is obtained comparable to one observed in the absence of Inc RNA. These results strengthen our previously proposed model that the pseudoknot induced by the translation and termination of the repY reading frame functions as the molecular switch for translational initiation of the repZ gene.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Evidence has been accumulating that RNA pseudoknots, stem-loop
structures containing an extended double helix with a complementary sequence outside the loop (for review, see Refs. 1 and 2), play an
important role in control of gene expression at the translational level. In the Escherichia coli ribosomal protein and
rpsO operons, RNA pseudoknots act as the binding sites for
proteins S4 and S15, respectively, being responsible for autoregulation
of the respective operon genes (3, 4). In some mammalian retroviruses
and in the mRNA for the rat ornithine decarboxylase antizyme, RNA pseudoknots function as regulatory signals for +1 or
1 frameshifting or translational read-through to allow translation of sequences downstream of stop codons (5-7). In IncI
ColIb-P9 and IncB pMU720 plasmids, possible RNA pseudoknots have been proposed to be the molecular switches for translation initiation of the replication initiator genes, thereby controlling plasmid copy number in the host
cells (8-10).
ColIb-P9 is a low-copy number and self-transmissible plasmid with a
size of 93 kilobases (kb).1
The basic replicon of ColIb-P9 consists of a 3-kb DNA fragment which
contains sufficient information required for autonomous replication and
copy number control (Ref. 11, also see Fig. 1). The frequency of ColIb-P9 replication
is limited by the degree of expression of the repZ gene
encoding a 39-kDa replication initiation protein, RepZ, which is
thought to react with the replication origin (11). Previous studies
revealed that repZ expression was negatively controlled by
the antisense Inc RNA of about 70 bases, the product of the
inc gene that governs the phenotype of plasmid
incompatibility (11, 12). Inc RNA is transcribed from the noncoding
strand of the leader region of repZ and binds to RepZ
mRNA at the complementary region to form an RNA-RNA duplex (12).
Two promoter down mutations, inc1 and inc2,
altered the 35 and
10 regions of the inc gene promoter,
pinc, and increased the level of repZ expression
without affecting significantly the amount of RepZ mRNA (Refs. 11
and 12; see Fig. 1B for positions of inc1 and
inc2). Thus, Inc RNA negatively controls repZ
expression at the translational level.
|
In an effort to elucidate the molecular events required for repZ expression, we also isolated replication-defective (rep) mutations (8, 13). Two types of rep mutations were isolated that were located in the leader region of repZ. One type including rep57 disrupted an upstream open reading frame called repY which encodes a polypeptide of 29 amino acids. rep57 was an amber mutation of repY codon-11 (13) and reduced the level of repZ expression without affecting the amount of RepZ mRNA (8, 13). Changing the amino acid composition of the reading frame by frameshift mutations did not affect repZ translation, whereas repZ translation was abolished profoundly when the position of the repY stop codon, located 7 bases downstream of the repZ start codon, was shifted either by frameshift mutations or changing the stop codon to a sense codon, indicating that the translational termination event of the repY reading frame, not the RepY polypeptide itself, is required for repZ translation (9). Since the level of repY expression was inversely correlated with the amount of endogenous Inc RNA (13) and the 5'-end of Inc RNA was located 3 bases upstream of the repY RBS (12), we concluded that inhibition of repY translation by Inc RNA is at least one mechanism by which Inc RNA represses repZ translation (13).
Surprisingly, however, the other type of rep mutations were
found to disrupt an intramolecular base pairing within RepZ mRNA between the 5'-rGGCG-3' and 5'-rCGCC-3' sequences, distantly separated at positions 327-330 and 437-440, respectively (Ref. 8, see Figs.
1B and 2C). rep2006 (G327A) and
rep2041 (G330A) changed the first and forth guanine residues
of the former, whereas rep2044 (G438A) altered the second
guanine residue of the latter (8). Besides, compensatory base changes
to each of the rep mutations in the other sequence restored
the ability to produce RepZ (8). This intramolecular base pairing had
the potential to form a pseudoknot with a stem-loop structure
designated I which was predicted as the target site of Inc RNA. On the
other hand, another stem-loop structure designated III sequestered the
5'-rCGCC-3' sequence together with the repZ RBS. Based on
these findings, we proposed a regulatory model (Fig. 1B), in
which translation and termination of repY induces the
intramolecular base pairing, leading to form a novel RNA pseudoknot for
repZ translation. Inc RNA inhibits the formation of the
pseudoknot, both directly and indirectly by inhibiting repY
translation. Consistent with this model, disruption of structure III by
two mutations (G451 and C459A) resulted in partial derepression of
repZ expression (9). In addition, Inc RNA inhibited
repZ expression more efficiently than repY
expression (8). However, biochemical evidence for the proposed
pseudoknot had not been provided.
Several other fundamental questions have arisen during the course of studies on the control of repZ translation: does pseudoknot formation simply expose the repZ RBS to the ribosome, or also enhance repZ translation through specific interaction with the ribosome? And why does a single base substitution in the proposed 5'-rGGCG-3'/5'-rCGCC-3' duplex affect profoundly the intramolecular base pairing, when the complementary sequences consist of 7 bp or more (see Fig. 2C)? What is the efficiency with which repY translation stimulate the initiation of repZ translation? To answer some of these questions, we set out to characterize biochemically the pseudoknot. In this report, we demonstrate that RepZ mRNA synthesized in vitro formed structures I and III, but not the pseudoknot. However, disruption of structure III by means of base substitution/deletion resulted in the formation of a unique RNA pseudoknot in vitro. Evidence is also presented indicating that the pseudoknot formed in vitro is generated in vivo, affecting directly the level of repZ expression.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bacteria, Phages, and Plasmids--
The E. coli K12
strains W3110 and W3110(ind
) were used as
the hosts of
:ColIb-P9 hybrid phages. Strains MV1184 (14) and NM522
(15) were the hosts of phage M13. Strains BW313 (dut ung) (16) and
BMH71-18mutS (17) were used for site-directed mutagenesis of the RepZ
mRNA leader region. Strain MC1061 (lacX74) (18) was
employed for lacZ fusion studies.
:ColIb-P9 hybrid phages and plasmids used in this study were listed in Table
I.
W3 (inc1) was isolated
as a clear-plaque forming mutant from
CH10W by plating with W3110
(
ind
) cells as described (11). The original
mini-ColIb-P9 replicon pCH10 (11) was found to have a copy-up mutation
by changing cytosine to adenine at position 334 (C334A) within the
inc gene region. Therefore, we re-constructed the wild type
mini-ColIb-P9 as pAK10 (22). pDX14-A25 (inc1 rep57 A25) and
pDX14-A28 (inc1 rep57 A28) were prepared by site-directed
mutagenesis as described (8) using oligo-A25,
5'-GTATTCTTCAGATTTTTCACTTTTTGTCGCTTATGG-3' and oligo-A28,
5'-CGCTTATGGCGGAGTTGTGCCGTGGTATT-3', respectively. pDX14-A33 was
constructed by introducing rep57, an amber mutation of
repY codon-11 (13), into pDX14-W3. pKA340-W3 was a
translational repZ-lacZ fusion and was used to measure the
level of repZ translation activity in the absence of Inc
RNA. pKA340-A52 carrying a repY-repZ-lacZ fusion in-frame
was constructed by insertion of a cytosine between positions 403 and
404 into pKA340-W3.
|
Measurement of -Galactosidase Activity--
The
-galactosidase activity expressed from translational lacZ
fusions was assayed as described (8). The specific activity of the
enzyme was expressed as Miller units (23). 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-- RNA corresponding to the RepZ mRNA leader region was synthesized in vitro using T7 RNA polymerase. As the template we used SmaI-digested pKA10, pKA10-A25, and pKA10-A28 for wild-type RNA293, A25 RNA293, and A28 RNA290, respectively, EcoO109I-digested pKA10 for RNA120, and NdeI-digested pKA16 and pKA16-A10 for RNA206 and rep2044 RNA206, respectively. SmaI cleaves pKA10 and its derivatives at the multiple cloning site of the vector, 3' to the ColIb-P9 insert, whereas EcoO109I cleaves in the middle of the ColIb-P9 portion, 3' to the structure I region (see Fig. 1). NdeI cleaves pKA16 or its rep2044 derivative at the NdeI site introduced in the middle of the structure III region of ColIb-P9 (Table I).
For preparation of the 5'-end-labeled products, the reaction mixture of 50 µl contained 40 mM Tris-HCl (pH 8.0), 6 mM MgCl2, 5 mM dithiothreitol, 2 mM spermidine, 2 mM each of ATP, CTP, and UTP, 0.5 mM GTP, 4.625 MBq of [RNase Digestion-- The 5'-end-labeled RNA (0.1 µg/reaction) was digested with serially diluted RNase T1 (Amersham Pharmacia Biotech), RNase derived from Bacillus cereus, RNase Bc (Amersham Pharmacia Biotech), or RNase V1 (Amersham Pharmacia Biotech) in 6 µl of the standard binding buffer in the presence of 0.5 µg of yeast tRNAs. After incubation for 10 min at 37 °C, the reaction was stopped by adding 4 µl of stop solution (95% formamide, 0.1% xylene cyanol, and 0.1% bromphenol blue) and placed on ice. 3 µl of the sample was resolved by electrophoresis on a 8.3 M urea, 8% polyacrylamide gel.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Secondary Structures of RepZ mRNA Leader Region-- To characterize biochemically secondary structures in the RepZ mRNA leader region, we prepared in vitro RNA293 labeled with 32P at the 5'-end as described under "Experimental Procedures." This RNA corresponds to the wild-type ColIb-P9 sequence from positions 244 to 524 that covered the entire region containing structures I to III. After the RNA sample was partially digested by RNases T1 (specific for guanine residues in single-stranded regions), RNase Bc (specific for pyrimidines in single-stranded regions), or RNase V1 (specific for double-stranded regions), the resulting products were analyzed by electrophoresis on a denaturing polyacrylamide gel. The patterns of the autoradiography are shown in Fig. 2, A and B, and the cleavage sites identified are summarized in Fig. 2C, in which the deduced secondary structures are also presented. We observed three stem-loop structures, I, II, and III as predicted previously (13). In addition, we found three new stem-loop structures designated Ia, IIa, and IV, which could not be predicted computationally. Note that RNase Bc appeared to cleave any ribonucleotides in the 5' side of loop sequences in the assay condition employed in this study.
|
In Vitro Formation of a Pseudoknot Structure-- On the basis of the above results, we disrupted structure III by substituting 8 bases of its 3' side sequence, 5'-rGCUUGUGGCAGG-3', to adenine residues without changing the repZ RBS (the substituted bases are underlined in the sequence). The resulting multiple mutation was designated as A25. Secondary structures of A25 RNA293 synthesized in vitro were analyzed in conjunction with mutation rep2041 (G330A) and rep2044 (G438A) defective in the intramolecular base pairing (8). When compared with the wild-type RNA293, RNase T1 sensitivities at G-327 and G-328 in A25 RNA293 were substantially reduced, although the cleavage pattern of the sequence corresponding to the 3' side of the structure I stem was not changed (A25 in Fig. 3A). Furthermore, the RNase V1 sensitivity at C-440 disappeared, indicating that structure III of A25 RNA293 was indeed disrupted by the base changes. On the other hand, when mutation rep2041 or rep2044 was introduced into A25 RNA293, the degree of the RNase sensitivities of G-327 and G-328 was restored to that observed in the wild-type RNA293 (A26 and A32 in Fig. 3A). Since C-329 and G-330 had been shown to interact with G-438 and C-437 by base pairing, respectively (8), these results can be explained by suggesting that, in A25 RNA293, at least 5'-rGGCG-3' in the loop of structure I base pairs with its complementary sequence 5'-rCGCC-3' located in the unfolded structure III region to form an RNA pseudoknot as shown in Fig. 3C (panel A25).
|
A Pseudoknot Formed in RNA206-- The sequence of the wild-type RepZ mRNA leader region showed that 10 out of 11 bases of the 5'-rUGGCGGAACGA-3' sequence (positions 326-336) in structure I were complementary to the downstream sequence just preceding the repZ RBS. Since A28 RNA290 deleted the 3 complementary bases from the sequence, we examined whether these bases also contributed to the pseudoknot formation. For this, we prepared RNA206, a deletion of RNA293 beyond A-443 (see Fig. 2C for the position of 3'-end of RNA206). This RNA lacked the sequence responsible for structure III, IIIa, or IIIb formation. When RNA206 was digested by RNases, the cleavage pattern of the structure I region was found to be quite different from that of the wild-type RNA293. (i) Both G-327 and G-328 were not cleaved by RNase T1; (ii) instead, G-321 was strongly cleaved by RNase T1; and (iii) RNase Bc sensitivity at U-317 was lost (Fig. 4A, lanes 1-3). However, the cleavage patterns of the sequence corresponding to the stem region in structure I were identical to those of RNA293 (Fig. 4A). Surprisingly, when rep2044 was introduced into RNA206, the cleavage patterns of structure I region became identical to those of RNA293 (Fig. 4A, lanes 4-6, and also see, Fig. 2B, lanes 1-3). These results indicate strongly that the 10 complementary bases in the top region of structure I had the potential to pair with those in the downstream sequence, generating a unique RNA pseudoknot as shown in Fig. 4B. Of further significance was the finding that this seemingly strong base pairing was abolished by introduction of a single mismatch mutation, rep2044. This phenomenon will be studied later in relation to the role of each base pair in repZ expression.
|
Effect of the Pseudoknot on repZ Expression--
To assess the
pseudoknot formed in vitro, we examined effects of mutations
A25 and A28 on the level of repZ expression in the absence
of inc and repY genes, the negative and positive
regulatory elements, respectively, as the action of either or both of
them might cause conformational changes in the RepZ mRNA leader
region (see Fig. 3C for effects of A25 and A28 mutations on
RepZ mRNA secondary structure). The RepZ activity was monitored by
measuring the -galactosidase activity of the repZ-lacZ
translational fusions, in which codon 221 of the repZ gene
was fused in-frame to codon 7 of the promoter-less lacZ gene
as the reporter. The vector for this fusion gene was a mini-F plasmid,
whose copy number was almost the same as that of ColIb-P9 (data not
shown). A summary of the experiments is given in Table
II. pKA340-W3 carrying inc1, a
promoter-down mutation in the inc gene, showed 1,912 units
of RepZ activity. When rep57 (C408T), an amber mutation of
repY codon 11 (13), was introduced into pKA340-W3, the
resultant plasmid pKA340-A33 exhibited only 2.6 units of the RepZ
activity, a value 735-fold (1912/2.6) lower than that of pKA340-W3. In
the case of pKA340-A25 carrying the A25 mutation, the level of RepZ
activity was 223 units, 86-fold higher than that of pKA340-A33. This
level was further elevated to 3,714 units in pKA340-A28 bearing the A28 mutation. The value of the RepZ activity in pKA340-A28 was twice as
high as that of pKA340-W3. There results indicate that disruption of
structures III and IIIa leads to a higher level of repZ
expression.
|
Bases Involved in Pseudoknot Formation in Vivo-- In view of the finding that all the base pairs in the pseudoknot of RNA206 were abolished by a single mismatch mutation, rep2044, we considered the role of each base pair in the pseudoknot formation. To address this problem and to characterize further the pseudoknot formed in vivo, the complementary sequences of pKA340-W3 were changed one by one and examined for repZ expression in the absence of Inc RNA. The results of the experiments are given in Table III. All the A:C mismatches in base pairings between 5'-rUGGCGG-3' (326-331) and 5'-rCCGCCA-3' (436-441) reduced repZ expression to various extents, and the most severe reduction was observed at the four middle contiguous base pairs, 5'-rGGCG-3'/5'-rCGCC-3', consistent with previous observations (8). On the other hand, mismatches of U:U or A:A at positions 332 and 435 and of A:G at positions 334 and 433 did not cause a significant reduction in repZ expression. Similarly, we observed that substitution of U-325 to C located outside the complementary sequence also did not affect the level of repZ expression. These results, taken together, indicated that 6 specific contiguous base pairings between 5'-rUGGCGG-3' (positions 326-331) and 5'-rCCGCCA-3' (positions 436-441) were sufficient for the pseudoknot to be formed in vivo.
|
Requirement for repY Translation in the Induction of repZ
Expression--
We have previously shown that the translation and
termination of the repY reading frame is essential for
repZ translation, and that Inc RNA regulates the translation
of both repY and repZ at different rates (8). In
this paper, the role of repY translation in repZ
expression was shown to involve disruption of structure III, inducing
pseudoknot formation (Table II). To investigate the effect of
repY translation on repZ expression, we
compared -galactosidase activities between pKA340-W3 and pKA340-A52,
carrying repZ-lacZ and repY-repZ-lacZ fusions in
mini-F derived plasmids, respectively, where in both cases the
lacZ gene was connected to the same position in the
repZ gene. The repY-repZ-lacZ fusion in
pKA340-A52 was constructed by inserting a cytosine residue between
positions 403 and 404 and copy number of this plasmid was the same as
that of pKA340-W3. Fig. 5 shows the
results of the experiments. pKA340-A52 exhibited 2151 units, a value
almost identical to that of pKA340-W3. In addition, we had previously shown that no protein synthesis was initiated from the normal repZ initiation codon in the repY-repZ-lacZ
fusions because of the lack of the termination event near the
repZ initiation codon (8, 9, 13). Assuming that the
-galactosidase activity of the repY-repZ-lacZ fusion
protein was not affected by the extra 25 amino acids derived from the
repY frame, these results indicated that the ratio of
repY translation to that of repZ translation was
nearly one, implying that ribosome access to the repZ RBS and the subsequent steps in initiation of repZ translation
are very efficiently coupled to repY translation and
termination.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this report, we have presented evidence that the novel RNA pseudoknot formed in the RepZ mRNA leader region is involved in translation initiation of the repZ gene, encoding the replication initiator protein of the ColIb-P9 plasmid. Structural analyses of the RepZ mRNA leader sequence synthesized in vitro revealed that it contained several stem-loop structures including structures I and III, but not the pseudoknot (Fig. 2). However, disruption of structure III, without changing the repZ RBS, by means of base substitution and deletion was found to induce new intramolecular base pairing between two distantly separated short complementary sequences located at positions 327-330 and 437-440. These interactions result in formation of a pseudoknot involving structure I (Fig. 3). We have also presented evidence suggesting that the pseudoknot observed in vitro is formed in vivo, affecting significantly the level of repZ expression (Table II). These results strengthen the previously proposed model that the pseudoknot induced by translation and termination of the repY reading frame functions as the molecular switch for translational initiation of the repZ gene.
Is the disruption of structure III sufficient for pseudoknot formation? The leader regions of the replication initiator (rep) genes of two plasmids, IncB pMU720 and IncK pMU2200, are similar in sequence to the corresponding region of ColIb-P9 (26). These three plasmids commonly have two stem-loop structures, one as the target site of the antisense RNA, and the other that sequesters the rep RBS. Pseudoknots with the target stem-loop for antisense RNA binding can also be predicted, and evidence for one in pMU720 is reported (10). However, no other stem-loop analogous to structure II of ColIb-P9 is predicted in pMU2200, and in fact, a 30-base long DNA segment encompassing structure II was able to be deleted without affecting the pseudoknot formation in ColIb-P9.2 In addition, we previously constructed an IncFII R100:ColIb-P9 chimeric mini-plasmid where the ColIb-P9 repZ leader sequence upstream of 5'-rCGCC-3' was replaced with the corresponding region of a distantly related R100 plasmid containing its entire inc gene plus the initiation codon of repA6 in-frame to the truncated ColIb-P9 repY frame (22). The leader regions of rep genes of IncFII-type plasmids encode an antisense RNA and upstream open reading frame such as inc and repA6 in R100, respectively, but the rep genes of these plasmids do not require formation of a pseudoknot for coupling translation of upstream open reading frame to rep (26). Although the resultant chimeric plasmid did not produce repZ due to lack of the pseudoknot structure, the replacement of the Inc RNA-target loop of the R100 portion with that of ColIb-P9, including the 5'-rGGCG-3' sequence, was sufficient for repZ in the ColIb-P9 portion to be translated (22). These lines of evidence together suggest that only structure I and the exposed 5'-rCGCC-3' sequence are sufficient for the pseudoknot formation, and hence repZ expression.
If so, how does the loop of structure I base pair specifically with a sequence preceding the repZ RBS to form a pseudoknot? And why does a single mismatch mutation such as rep2044 disrupt it profoundly, although possible base pairing occurs between 11 bases of complementary RNA sequences (Fig. 4)? We believe that a transient base paring between a subset of these sequences, 5'-rGGCG-3' and 5'-rCGCC-3', is rate-limiting for the formation of the pseudoknot. Thus, interfering with this transient step by a single mismatch can be sufficient for preventing all subsequent steps in pseudoknot formation. In addition, a certain conformation of the loop region of structure I appears to play a role in stimulating the intramolecular base pairing by giving it higher affinity and specificity, as suggested here by the preference for a pyrimidine residue at a base 5' to the 5'-rGGCG-3' sequence for repZ expression (Table III). The same conformation in the loop of structure I may stimulate RepZ mRNA-Inc RNA interaction as well. Detailed analyses of the structural basis of the intermolecular interaction in the accompanying paper (30), as well as the analyses of the pseudoknot formation in vitro with different RNA species that inhibit binding of Inc RNA to the RepZ mRNA leader3 support this hypothesis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Sota Hiraga for providing pXX564, Shigeyuki Yokoyama for the gift of purified T7 RNA polymerase, and Alan G. Hinnebusch and Robert Weisberg for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported 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. Tel.: 301-594-7240; Fax: 301-496-8576. E-mail: kasano{at}aghmac1.nichd.nih.gov.
1 The abbreviations used are; kb, kilobase(s); bp, base pair(s); RBS, ribosome-binding site; Kmr, kanamycin resistant; RNase, ribonuclease.
2 K. Asano and K. Mizobuchi, unpublished observations.
3 K. Asano and K. Mizobuchi, manuscript in preparation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|