Structural Basis for Binding of the Plasmid ColIb-P9 Antisense Inc RNA to Its Target RNA with the 5'-rUUGGCG-3' Motif in the Loop Sequence*

Katsura AsanoDagger §, Tatsuya NiimiDagger , Shigeyuki YokoyamaDagger , and Kiyoshi Mizobuchi

From the Dagger  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
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Abstract
Introduction
Procedures
Results
Discussion
References

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 IncIalpha 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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 IncIalpha 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 IncIalpha 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.

    EXPERIMENTAL PROCEDURES
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Introduction
Procedures
Results
Discussion
References

Bacteria, Phages, and Plasmids-- Escherichia coli K12 strains used in this study were W3110, W3110lambda ind-, 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. lambda :ColIb-P9 hybrid phage lambda CH10W was constructed by subcloning the 3.0-kilobase EcoRI fragment of pAK10 into the EcoRI site of lambda VIII (20). lambda W1 is a spontaneous copy-up mutant of ColIb-P9 isolated from lambda CH10W (see below).

                              
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Table I
List of plasmids used in this study

Isolation of Copy-up Mutants Using lambda :ColIb-P9 Hybrid System-- The procedure for isolation of copy-up mutants using lambda :ColIb-P9 hybrid was described (7). As the original lambda :ColIb-P9 hybrid, lambda 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 lambda :ColIb-P9 hybrid phage, lambda CH10W. Clear-plaque forming mutants of lambda CH10W plated on W3110lambda 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 lambda 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 lambda 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 beta -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 beta -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 [gamma -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.

32P-Labeled Inc RNA was synthesized using the supercoiled pKA10 DNA as a template and E. coli RNA polymerase. The reaction mixture of 50 µl contained 40 mM Tris-HCl (pH 7.6), 150 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 0.02 mg/ml bovine serum albumin, 500 mM each of ATP, CTP, GTP, and UTP, 2.96 MBq of [alpha -32P]UTP (222 TBq/mmol), 40 units of RNase inhibitor (Takara Shuzo, Kyoto), 6 units of E. coli RNA polymerase (Boehringer Manheim), and 5 µg of plasmid DNA. Inc RNA labeled with 32P at the 5' end was synthesized in the same reaction mixture (total of 80 µl) as for uniformly labeled ones except for 300 µM cold GTP and 3.7 MBq of [gamma -32P]GTP (222 TBq/mmol) instead of 500 µM cold GTP and 2.96 MBq of [alpha -32P]UTP (222 TBq/mmol). For mutant derivatives of 32P-Inc RNA (both 5'-labeled and uniformly labeled), we used mutant derivatives of pKA10 as the template. All the radioactive materials were purchased from New England Nuclear Research Products. These RNAs were gel-purified (23) and suspended in the standard binding buffer of Tomizawa (20 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 100 mM NaCl) (24). Concentrations were determined by scintillation counting.

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 × 10-10 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).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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 lambda W1, derived from the lambda :ColIb-P9 hybrid phage, lambda CH10W (see "Experimental Procedures"). Copy number of the ColIb-P9 replicon excised from lambda W1 was increased 36-fold compared with wild-type (data not shown). repZ expression was increased 75-fold by T325C when measured by beta -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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Fig. 1.   RNA-RNA interactions involved in control of plasmid ColIb-P9 repZ gene expression. RepZ mRNA and Inc RNA are depicted with nucleotide sequences in the relevant portion. Inc RNA is a negative regulatory element for repZ expression, whereas a pseudoknot structure, involving stem-loop structure I and two short complementary sequences at positions 327-330 and 437-440 (boxed), is a positive regulatory element for repZ expression. Both interactions, shown by dotted arrows, involve the conserved 5'-rUUGGCG-3' sequence in structure I (boxed with dotted line). The position of stem-loop structure III is shown by arrows. The 5'-rCGCCAA-3' sequence in Inc RNA, complementary to the hexanucleotide sequence, is also boxed with a dotted line. See the accompanying paper (11) for details on mechanism of induction of the unique pseudoknot structure. Locations and base changes of mutations used in this study are indicated by arrows. The start codons of repY and repZ are shown by hooked arrows.

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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Table II
Effect of mutations in the inc gene region on repZ gene expression
MC1061 cells carrying indicated combinations of the Inc RNA-donor and recipient repZ-lacZ fusion plasmids were grown to A600 = 0.4 and assayed for beta -galactosidase activity as described under "Experimental Procedures." pACYC184 (18) is a vector and does not carry the inc gene.

To examine the effect of different mutant Inc RNAs on repZ expression, the vector pACYC184 was replaced with pKA18 or its mutant derivatives encoding wild-type or mutant Inc RNAs, respectively. As shown in Table II, each of the reporter constructs exhibited repZ expression ranging from 1.2 to 42 units in the presence of the mutant Inc RNA with the complementary substitution (Table II, the second line in each mutant subset). As these values are significantly higher than 0.3 unit from the wild-type combination (line 2, column 5), we concluded that all the mutations employed here affected the action of Inc RNA, even though the two interacting RNA molecules are complementary to each other.

Next, we replaced either the recipient or donor plasmid with a wild-type one. In these heterologous combinations, base pairing between Inc RNA and RepZ mRNA at the mutation site was mismatched as shown in column 4, Table II. We found the effect of replacement (or mismatch) differed by the mutation site. Accordingly, each mutation was classified into two types. In the type I mutations such as rep2006, G328A, and C329T, a C:A mismatch at the mutation site resulted in the highest level of repZ expression in the presence of Inc RNA between 55 and 124 units (lines 21, 26, and 29). The fact that the type I mutations were located in three contiguous bases (5'-rGGC-3'/3'-rCCG-5') at position 327-329 suggest strongly that an initial base pairing interaction between Inc RNA and structure I at these positions is rate-limiting for the intermolecular interaction in vivo. On the other hand, the decreased action of Inc RNA with T325C, T326C, T326G, or G331A mutations on the repZ-lacZ reporter plasmid carrying complementary substitution was partially overcome by replacing either the donor or recipient plasmid with wild-type (lines 5, 9, 14, and 34). We classified these mutations as type II. We reasoned that the decreased action of Inc RNA in the complementary donor-recipient combination of these mutants was due to a requirement for specific Inc RNA or RepZ mRNA sequence (structure) in addition to ability to base pair.

The G330A mutation, also known to be as defective in pseudoknot formation as rep2041 (9), did not belong to either of these classes, since any replacement did not affect repZ expression (Table II). The same holds true for the moderate copy-up mutation, C334A. As will be described later, however, these mutations could be classified as type I' (a subtype of type I) and type II, respectively, by assaying in vitro binding between Inc RNA and RepZ mRNA (see below).

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 × 10-10 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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Fig. 2.   Hybridization between Inc RNA and RepZ mRNA in vitro. A, autoradiography of formation of the Inc RNA-RepZ mRNA hybrid. The three panels show the hybridization between 32P-labeled wild-type Inc RNA and unlabeled wild-type RNA293, between 32P-labeled rep2006 Inc RNA and unlabeled rep2006 RNA293, and between 32P-labeled C334A Inc RNA and unlabeled C334A RNA293, respectively. In vitro preparation of Inc RNA and RNAs293 and conditions of the experiments are described under "Experimental Procedures." The positions of Inc RNA and the hybrid formed are indicated as Inc RNA and Hybrid, respectively, in the figure. The position of free RNA293 determined by electrophoresis of 32P-labeled product is indicated by arrows. B, kinetic analyses of binding of 32P-labeled Inc RNA to RNA293. Ratio of free and bound Inc RNA was determined and plotted by measuring the intensity of the autoradiograms using a Shimadzu CS-930 scanner.

When the same amount of RNA293 and Inc RNA, each carrying rep2006 and the complementary base change, respectively, were incubated together, the apparent binding rate decreased as shown in Fig. 2B, confirming in vitro that the binding of Inc RNA was impaired by the replication-defective mutation. The same was true for the moderate copy-up mutation, C334A (Fig. 2B). Likewise, the binding rate constants for the complementary pairs of mutant Inc RNAs and RNAs293 were measured and shown in the first line of each mutant subset in Table III. As a result, we found that all the base substitutions reduced the intermolecular base pairing. To determine which RNA caused the reduction of binding rate constants, Inc RNA or RNA293, we performed the binding experiments using the non-complementary pairs, one of which was the wild-type Inc RNA or RNA293 (the second and third lines in each mutant subset in Table III). As observed in vivo, the mutations were classified into two types, but the division here was even more dramatic (see below). For the type I mutations including rep2006, G328A, and C329T, the rate constants decreased in the order of stability of canonical base pairing (G:C > A:U > G:U > A:C) between Inc RNA and RNA293 at the mutation site, indicating that the base pairing between 5'-rGGC-3' (RNA293) and 3'-rCCG-5' (Inc RNA) at positions 327-329 was critical also for the intermolecular interaction in vitro. For the G330A mutation that could not be classified into either of the two in vivo, the rate constant in the case of C:A pair was the lowest, although it was close to the value obtained with the G:U pair. Therefore, we classified G330A as a subtype of type I, designated as type I'.

                              
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Table III
Effect of the inc loop mutations on hybridization between Inc RNA and RepZ mRNA leader
Wild-type or mutant 32P-Inc RNA was hybridized with wild-type or mutant RNA293 in the indicated combinations, as described under "Experimental Procedures." Rate constants of hybridization were determined and presented as relative values to the wild-type pair; 6.8 × 105 /M /s.

For the type II mutations, the rate constants were increased by restoring the altered nucleotide in either Inc RNA or RNA293 to the wild-type base (Table III). These results indicate that the reduced binding rates in the type II mutations were due to a lesion in the RNA component whose mutation was restored. Mutations of type II were further subclassified according to which component, Inc RNA or RNA293, was more affected by each mutation. The reductions by T326G and G331A were attributed to change in RNA293 (type IIa), whereas the reductions caused by T325C, T326C, or C334A mutation were mainly due to alterations in Inc RNA (type IIb). Thus, the overall pattern of the effects of mutations on in vitro binding reflected repZ-lacZ expression in vivo, although the binding rate constants (Table III) did not correlate one-to-one with repZ expression (Table II). This could be due to differential effects of the mutations on metabolic stability of RepZ mRNA or Inc RNA in vivo as well as differences in reaction condition.

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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Fig. 3.   Stem-loop structure I in wild-type and mutant RepZ mRNAs. A, cleavage patterns of wild-type and mutant RepZ mRNA. Wild-type and mutant 32P-RNA120 was partially digested with RNases T1, Bc, and V1 and analyzed as described (11) (lanes 1-3, respectively). The length of RNase T1 cleavage products in wild-type RNA120 and the corresponding residues with ColIb-P9 coordinates (in boldface in parentheses) were shown by the side of the products. For mutant RNA120, the products whose cleavages are enhanced or reduced are indicated by filled or open triangles, respectively. The size of triangles indicates intensities of cleavage. The residues and ColIb-P9 coordinates are written in boldface. B, secondary structures deduced from the patterns shown in A. RNA sequences from position 302 to 353 of RNA120 are depicted. The changed bases are highlighted. The bases where type I mutations were mapped are boxed. Filled, gray, and open triangles indicate the cleavage sites for RNases T1, Bc, and V1, respectively. The size of triangles indicates intensities of cleavage.

To know what kind of structural changes caused the reduction of the binding rate constants particularly with type II mutations, we next analyzed the secondary structures of the structure I regions for mutant RepZ mRNA using 5'-labeled 32P-RNA120 which contained a part of the RepZ mRNA leader from position 244 to 353, including structures Ia and I (11). The purified sample was partially cleaved with RNase T1, RNase Bc, or RNase V1 (specific for double-strand DNA), and sites of cleavage determined by gel electrophoresis. The RNase cleavage patterns of mutant RepZ mRNA is shown in Fig. 3A. In most of the mutants, degrees of cleavage by RNase T1 or RNase Bc at the upper loop domain of structure I were altered from wild-type, as judged by comparing them with degrees of cleavage at the single-stranded region upstream of structure I between C-288 and G-297 (see the cleavage products below G-297 in each panels in Fig. 3A). Based on these observations, we deduced secondary structures in mutant structure I as shown in Fig. 3B.

When we examined the effect of each type I or type I' mutation on the secondary structures of RNA120, RNase cleavage patterns were not changed compared with wild-type except for the loss of the RNase T1 sensitivity at position 327 in G327A and at position 328 in G328A due to the respective base changes of G to A (Fig. 3A). These results confirmed that the decrease in the binding rate constants by the type I and I' mutations were not due to the changes in secondary structures of either of the RNA components, but rather to the impaired base pairing interactions between them at the mutated positions.

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 [gamma -32P]GTP or [gamma -32P]ATP for labeling Inc RNA during the in vitro transcription reaction, it was labeled only with [gamma -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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Fig. 4.   Secondary structure analysis of wild-type Inc RNA. A, wild-type 5'-32P-Inc RNA was prepared as described under "Experimental Procedures" and partially digested with NaOH and RNases T1, Bc, and V1 (lanes 1-4, respectively). Left and right panels indicate the autoradiography of cleavage products analyzed by electrophoresis on denaturing 10 and 8% polyacrylamide gels, respectively. The length of RNase cleavage products and the corresponding residues with ColIb-P9 coordinates (in boldface in parentheses) were shown by the side of the products. B, secondary structure of Inc RNA. The RNA sequence of the in vitro synthesized Inc RNA is depicted with the 3'-ends shown by arrows. Numerals indicate the position of the bases in ColIb-P9 coordinate (see footnote 1). The bases where type I mutations were mapped are boxed. Filled, gray, and open triangles indicate the cleavage sites for RNases T1, Bc, and V1, respectively. The size of triangles indicates intensities of cleavage. The dotted line indicates weak base pairing.

Based on the overall cleavage patterns, we propose that Inc RNA is folded as depicted in Fig. 4B. We reasoned that the difference in RNase T1 sensitivity at G-320', G-324', and G-334' reflected different degrees of base pairing involving each of these residues. It is noteworthy that the upper domain of Inc RNA stem-loop appears to be folded less tightly than the complementary region in structure I.

We then analyzed the secondary structures of mutant Inc RNAs using RNases T1 and V1 (Fig. 5A). Changes in RNase T1 and V1 sensitivity were judged by normalizing the intensity of each cleavage product relative to the cleavage product(s) at G-358' and at U-340', U-341', and C-342', respectively. The sites of cleavage along with the deduced secondary structures were summarized in Fig. 5B. The type I and I' mutations did not change the RNase cleavage patterns of 5'-32P-Inc RNA, except that the strong RNase T1 cleavage at G-329' disappeared in C329T due to the substitution of G-329' to A in Inc RNA.


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Fig. 5.   Secondary structures in mutant Inc RNAs. A, cleavage patterns of wild-type and mutant Inc RNA. Wild-type and mutant 32P-Inc RNA were partially digested with RNases T1 and V1 and analyzed by polyacrylamide gel electrophoresis (lanes 1 and 2, respectively). The length of RNase T1 cleavage products in wild-type Inc RNA and the corresponding residues with ColIb-P9 coordinates (in boldface in parentheses) were shown by the side of the products. Weak RNase T1 cleavages at G-320' and G-334' observed in Fig. 4A are not obvious in this figure because of resolution. For mutant Inc RNA, the products whose cleavages are enhanced or reduced are indicated by filled or open triangles, respectively. The residues and ColIb-P9 coordinates (footnote 1) are written in boldface. B, secondary structures in wild-type and mutant Inc RNA. RNA sequences of wild-type and mutant Inc RNA are depicted. The changed bases are highlighted. The bases where type I mutations were mapped are boxed. Filled and open triangles indicate the cleavage sites for RNases T1 and V1, respectively. The size of triangles indicates intensities of cleavage. The dotted lines indicate weak base pairing.

Many of the type II mutations changed the cleavage patterns (Fig. 5A). T326G did not change the cleavage pattern as expected from its classification as type IIa (Fig. 5B and Table III, line 10). G331A reduced the RNase V1 sensitivity at G-334' and instead, weak RNase T1 cleavage occurred at the same position. In addition, the weak RNase T1 cleavage at G-334' was enhanced (Fig. 5A). These observations indicate that the upper loop domain of G331A Inc RNA was further destabilized by the base substitution affecting possibly the weak base pairing between C-331' and G-324' (Fig. 5B). However, the destabilization of the Inc RNA structure by G331A did not affect its binding ability as a type IIa mutation (Table III, line 25).

In contrast, T325C Inc RNA had a tighter upper loop domain as evidenced by decreased RNase T1 sensitivity at G-329' and G-324' (Fig. 5, A and B). T326C Inc RNA appeared to have an upper loop domain with altered secondary structure as shown in Fig. 5B, since the altered G residue at 326' was not cleaved by RNase T1 (Fig. 5A). The changes in secondary structures of T325C and T326C Inc RNAs were further supported by the faster mobility of these RNAs on an 8.3 M urea, 8% polyacrylamide gel than that of wild-type Inc RNA (data not shown). Furthermore, the secondary structure of C334A Inc RNA was also changed since the RNase V1 cleavage site was shifted (Fig. 5A, lane 2 in C334A). As the three mutations T325C, T326C, and C334A affected Inc RNA as type IIb mutations (Table III, lines 4, 7, and 28, respectively), creating any secondary structure in the upper loop domain of Inc RNA interferes with its binding to the target. Taken together with the results obtained with G331A Inc RNA, we conclude that the unstructured nature of the Inc RNA loop favors the intermolecular interaction with RepZ mRNA.

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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Table IV
Effect of RepZ mRNA pseudoknot on binding rate constants with Inc RNA
Wild-type 32P-Inc RNA was incubated with the indicated species of RepZ mRNA derivative and measured for the binding rate constant, as described under "Experimental Procedures."

Do Inc RNA binding and the pseudoknot formation function competitively in vivo as expected if they are mutually exclusive events? During the course of analyzing the copy number of mutant mini ColIb-P9 plasmids, we found that pA4, a derivative of pAK10 carrying rep2006 (Fig. 1), was maintained at a copy number slightly less than, but comparable to, the wild-type, suggesting that the reduced pseudoknot formation was compensated for by reduced Inc RNA binding in the mutant.2 To investigate how this mutant produced RepZ protein in amounts sufficient for replication, repY and repZ expression from the rep2006 mutant was analyzed further (Table V). Although RepZ activity of pKA340-A4 carrying only rep2006 was almost identical to that of the wild-type pKA340, RepY activity in rep2006 as measured by beta -galactosidase activity in a repY-repZ fusion was 12 times higher than that in the wild-type (compare pKA340-A63 with pKA340-A69), suggesting that many more rounds of repY translation were needed in the presence of rep2006 to stimulate a wild-type amount of repZ expression. The fact that RepZ activity of pKA340-A39 (rep2006 inc1) was reduced 4-fold by rep57 (in pKA340-A67) in the absence of Inc RNA confirmed that repZ expression depended on repY translation even in the presence of rep2006. Based on these results, we concluded that the reduced pseudoknot formation by rep2006 was compensated for by the elevated level of repY translation due to the decreased Inc RNA activity conferred by the rep2006 mutation. Thus, Inc RNA binding appears to function competitively with formation of the pseudoknot in vivo.

                              
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Table V
Analysis of repZ and repY expression from the rep2006 mutant

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 pi -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 pi -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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Fig. 6.   A model of loop conformation conferred by the 5'-rUUGGCG-3' motif within RepZ mRNA stem-loop I. In A, helical nature of 3' side of the anticodon loop of tRNA is illustrated schematically by the curved thick line representing the phosphate backbone. Bases in the loop are depicted by boxes and numbered as reported (28). Pyrimidine, uridine, and purine residues are conserved at positions 32, 33, and 37, respectively. Dotted line between boxes denote hydrogen bonding that closes the loop. This loop conformation is conferred by base stacking between residues in the helix and pi -turn of the backbone at U-33 (filled box), represented by bending of the thick line (26, 27). By analogy, we propose that the 5'-rUUGGCG-3' loop of ColIb-P9 RepZ mRNA structure I adopts similar helical conformation, as illustrated in B. Numbers in parentheses denote residues in the anticodon loop, employed for modeling the loop structure. In three-dimensional model, bases of Y-32 and R-37 of the anticodon loop come close, and if replaced with U and G, respectively, a wobble base pairing is possible, although weakly.


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Fig. 7.   The 5'-rUUGGCG-3' motifs in prokaryotic antisense control systems. A, RNA sequences of antisense and target RNAs with deduced secondary structures are described as reported previously (3, 4, 32). The 5'-UUGGCG-3' motif, highlighted by black boxes, is proposed as a structural motif to stimulate the initial base pairing interaction between the triple bases (shown by open boxes) in antisense and target RNAs. The names of plasmids and transposons and the genes controlled are shown below the RNA sequences. The antisense RNA I and its target RNA II of pMU720 plasmid of IncB group are very similar in sequence and structure to Inc RNA and stem-loop I of RepZ mRNA of ColIb-P9 (7-base substitutions and one base deletion located below G-335:C-320 pair of ColIb-P9 stem-loop I) (6, 33). B, stepwise models for binding between antisense RNA and its target with the 5'-rUUGGCG-3' motif in IncIalpha ColIb-P9, IncB pMU720, and possibly IncFII R1 plasmids. In each panel representing step of binding, antisense RNA (top) and its target (bottom) are described with hairpin-shaped lines denoting a single stem-loop structure. 5' and 3' ends of these molecules are indicated, and intermolecular base pairings are depicted by short bars. In a, the "S" shaped line in the upper loop domain of target RNA represents helical nature of the loop conferred by the 5'-rUUGGCG-3' motif, indicated by a bracket. Due to this conformation, the 5'-rGGC-3' sequence in the motif, represented by three protrusions, recognizes the complementary sequence in the loop of antisense RNA (b). Low thermodynamic stability of the loop structure allows this transient base pairing to propagate by a few more base pairs, thereby stabilizing the complex (c). Biochemical evidence for these transient steps were reported for IncFII R1 (31) and IncB pMU720 (6) systems and will be reported elsewhere for IncIalpha ColIb-P9.2 Following these steps, the complex irreversibly leads to formation of more stable complexes (d and e). For negative control of replication initiator (rep) expression, it has been proposed that complexes d and e are sufficient for repressing translation of upstream open reading frames, which is coupled to translation of the rep genes (6, 32). In IncIalpha ColIb-P9, complex c is stable enough to repress formation of the pseudoknot, which probably follows steps similar to steps a to c.2

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 IncIalpha 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.

    ACKNOWLEDGEMENTS

We thank H. Moriwaki for excellent technical assistance, and Alan Hinnebusch and Robert Weisberg for critical reading of the manuscript.

    FOOTNOTES

* 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.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Eguchi, Y., Itoh, T., and Tomizawa, J. (1991) Annu. Rev. Biochem. 60, 631-652[CrossRef][Medline] [Order article via Infotrieve]
  2. Wagner, E. G. H., and Simons, R. W. (1994) Annu. Rev. Microbiol. 48, 713-742[CrossRef][Medline] [Order article via Infotrieve]
  3. Couturier, M., Bex, F., Bergquist, P. L., and Maas, W. K. (1988) Microbiol. Rev. 52, 375-395
  4. Sugiyama, T., and Itoh, T. (1993) Nucleic Acids Res. 21, 5972-5977[Abstract]
  5. Kittle, J. D., Simons, R. W., Lee, J., and Kleckner, N. (1989) J. Mol. Biol. 210, 561-572[Medline] [Order article via Infotrieve]
  6. Siemering, K. R., Praszkier, J., and Pittard, A. J. (1994) J. Bacteriol. 176, 2677-2688[Abstract]
  7. Hama, C., Takizawa, T., Moriwaki, H., Urasaki, Y., and Mizobuchi, K. (1990) J. Bacteriol. 172, 1983-1991[Medline] [Order article via Infotrieve]
  8. Shiba, K., and Mizobuchi, K. (1990) J. Bacteriol. 172, 1992-1997[Medline] [Order article via Infotrieve]
  9. Asano, K., Kato, A., Moriwaki, H., Hama, C., Shiba, K., and Mizobuchi, K. (1991) J. Biol. Chem. 266, 3774-3781[Abstract/Free Full Text]
  10. Asano, K., Moriwaki, H., and Mizobuchi, K. (1991) J. Biol. Chem. 266, 24549-24556[Abstract/Free Full Text]
  11. Asano, K., and Mizobuchi, K. (1998) J. Biol. Chem. 273, 11815-11825[Abstract/Free Full Text]
  12. Vieira, J., and Messing, J. (1987) Methods Enzymol. 153, 3-11[Medline] [Order article via Infotrieve]
  13. Gough, J., and Marray, N. (1983) J. Mol. Biol. 166, 1-19[Medline] [Order article via Infotrieve]
  14. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 448-492
  15. Kramer, B., Kramer, W., and Fritz, H. J. (1984) Cell 38, 879-887[Medline] [Order article via Infotrieve]
  16. Casadaban, M. J., and Cohen, S. N. (1980) J. Mol. Biol. 138, 179-207[Medline] [Order article via Infotrieve]
  17. Mead, D. A., Szczesna-Skorupa, E., and Kemper, B. (1986) Protein Eng. 1, 67-74[Abstract]
  18. Chan, A. C. Y. C., and Cohen, S. N. (1978) J. Bacteriol. 134, 1141-1156[Medline] [Order article via Infotrieve]
  19. Kato, A., and Mizobuchi, K. (1994) DNA Res. 1, 201-212[Medline] [Order article via Infotrieve]
  20. Murry, N. E., and Murry, K. (1974) Nature 251, 476-481[Medline] [Order article via Infotrieve]
  21. Birnboim, H. C., and Doly, J. (1979) Nucl. Acids Res. 7, 1513-1523[Abstract]
  22. Miller, J. H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  23. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  24. Tomizawa, J. (1984) Cell 38, 861-870[Medline] [Order article via Infotrieve]
  25. Cohen, S. N. (1995) Cell 80, 829-832[Medline] [Order article via Infotrieve]
  26. Sussman, J. L., Holbrook, S. R., Wade Warrant, R., Church, G. M., and Kim, S.-H. (1978) J. Mol. Biol. 123, 607-630[Medline] [Order article via Infotrieve]
  27. Moras, D., Comarmond, M. B., Fischer, J., Weiss, R., Thierry, J. C., Ebel, J. P., and Giege, R. (1980) Nature 288, 669-674[Medline] [Order article via Infotrieve]
  28. Sprinzl, M., Hartmann, T., Weber, J., Blank, J., and Zeidler, R. (1989) Nucleic Acids Res. 17, (suppl.) R1-R172
  29. Jaeger, J. A., and Tinoco, I., Jr. (1993) Biochemistry 32, 12522-12530[Medline] [Order article via Infotrieve]
  30. Wilson, I. W., Siemering, K. R., Praszkier, J., and Pittard, A. J. (1997) J. Bacteriol. 179, 742-753[Abstract]
  31. Hjalt, T., and Wagner, E. G. H. (1995) Nucleic Acids Res. 23, 580-587[Abstract]
  32. Malmgren, C., Wagner, E. G. H., Ehresmann, C., Ehresmann, B., and Romby, P. (1997) J. Biol. Chem. 272, 12508-12512[Abstract/Free Full Text]
  33. Siemering, K. R., Praszkier, J., and Pittard, A. J. (1993) J. Bacteriol. 175, 2895-2906[Abstract]


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