Antisense RNA Control of Plasmid R1 Replication
THE DOMINANT PRODUCT OF THE ANTISENSE RNA-mRNA BINDING IS NOT A FULL RNA DUPLEX*

(Received for publication, January 31, 1997)

Charlotta Malmgren Dagger , E. Gerhart H. Wagner §, Chantal Ehresmann , Bernard Ehresmann and Pascale Romby par

From the Department of Microbiology, Biomedical Center, Uppsala University, Box 581 S-751 23 Uppsala, Sweden, § Department of Microbiology, Uppsala Genetic Center, SLU (Swedish University of Agricultural Sciences), Box 7025, Genetikvägen 5, S-75007 Uppsala, Sweden, and  UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue R. Descartes, Strasbourg cedex, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The replication frequency of plasmid R1 is controlled by an antisense RNA (CopA) that binds to its target site (CopT) in the leader region of repA mRNA and inhibits the synthesis of the replication initiator protein RepA. Previous studies on CopA-CopT pairing in vitro revealed the existence of a primary loop-loop interaction (kissing complex) that is subsequently converted to an almost irreversible duplex. However, the structure of more stable binding intermediates that lead to the formation of a complete duplex was speculative. Here, we investigated the interaction between CopA and CopT by using Pb(II)-induced cleavages. The kissing complex was studied using a truncated antisense RNA (CopI) that is unable to form a full duplex with CopT. Furthermore, RNase III, which is known to process the CopA-CopT complex in vivo, was used to detect the existence of a full duplex. Our data indicate that the formation of a full CopA-CopT duplex appears to be a very slow process in vitro. Unexpectedly, we found that the loop-loop interaction persists in the predominant CopA-CopT complex and is stabilized by intermolecular base pairing involving the 5'-proximal 30 nucleotides of CopA and the complementary region of CopT. This almost irreversible complex suffices to inhibit ribosome binding at the tap ribosome binding site and may be the inhibitory complex in vivo.


INTRODUCTION

Natural antisense RNAs control a variety of biological functions, mainly in prokaryotic accessory genetic elements such as phages, transposons, and plasmids (1). Most antisense RNAs found regulate the plasmid copy number. The efficiency of antisense RNA control depends on the rate constant for the binding between antisense and target RNA and on the intracellular concentration of the inhibitor RNA. The binding pathways were extensively studied in ColE1 (2-4) and R1 (5-7) plasmids. In both systems, the binding process is seen as a series of reactions leading to a stable duplex. The initial, rate-limiting step involves the formation of a transient loop-loop interaction (kissing complex) that is followed by a subsequent nucleation step via the single-stranded 5' segment of the antisense RNA. This interaction is believed to result in the formation of a double-stranded RNA along the entire length of the target site. However, the full duplex may not be required for inhibition since the transient kissing complex appears to be sufficient for control in ColE1 (8), R1 (9), and IncB (10) plasmids.

Plasmid R1 belongs to the IncFII group whose initiation frequency is controlled by an antisense RNA, CopA. CopA inhibits RepA synthesis by binding to the leader region of the repA mRNA (CopT) 80 nucleotides upstream of the repA translational start codon (11). RepA synthesis requires the translation of a short leader peptide (Tap) encoded immediately downstream of the CopA target site, overlapping the repA reading frame by two nucleotides (12). This implies that CopA inhibits repA translation indirectly by preventing tap translation in vivo (13). Extensive mutational analyses revealed that the structures of both the antisense and the target RNAs are critical for obtaining an optimal binding rate and in vivo control (1). However, although the role of loop-loop interactions in the binding pathway of the antisense RNA and its target has been characterized (7-9), the structure(s) of more stable binding intermediate(s) that lead to a complete duplex remained speculative. In the present study, the interaction between CopA and CopT was followed by using Pb(II)-induced cleavages. The loop-loop interaction was also probed using a truncated antisense RNA (CopI) that is unable to form a full duplex with CopT (6). Furthermore, RNase III that processes the CopA-CopT complex (14) was used to distinguish between antisense-target RNA complexes differing in the extent of duplex formation. Our results indicate that the stable CopA-CopT complex is not fully base-paired in the region of complementarity. Remarkably, an extended loop-loop interaction persists in the CopA-CopT complex, maintaining the structures of the major CopA and CopT stem loops. This kissing complex is additionally stabilized by an intermolecular helix involving the 5'-proximal 30 nucleotides of the antisense RNA and its counterpart in CopT. The significance of this CopA-CopT complex for the regulatory mechanism is discussed.


EXPERIMENTAL PROCEDURES

DNA Templates and RNA Synthesis

CopT, CopA, and CopI were synthesized with T7 RNA polymerase from polymerase chain reaction-generated DNA fragments as described by Hjalt and Wagner (15). Transcription of CopT yields a run-off product of 302 nucleotides that carries two G residues at its 5' end instead of the GU sequence of the wild type repA mRNA. The CopA RNA contains a 5'-terminal G instead of an A residue. 5'-End labeling of dephosphorylated RNA was performed with T4 polynucleotide kinase and [gamma -32P]ATP (16). Labeled RNAs were purified by polyacrylamide-urea gel electrophoresis, eluted, and precipitated twice with ethanol. Before use, the different RNAs were dissolved in RNase-free water and renatured by incubation at 90 °C for 2 min followed by slow cooling at room temperature in TMN buffer (20 mM Tris acetate, pH 7.5, 10 mM magnesium acetate, 100 mM sodium acetate).

Pb(II)-induced Cleavages

Experimental conditions were adapted from Hjalt and Wagner (15). Complex formation was carried out at 37 °C for 5 or 15 min in TMN buffer with 5'-end-labeled CopT (3 × 10-8 M) and a 5-fold excess of unlabeled CopA or CopI (1.5 × 10-7 M) or with 5'-end-labeled CopA or CopI (4 × 10-8 M) and a 5-fold excess of unlabeled CopT (2 × 10-7 M). Full duplexes between 5'-end-labeled CopT and CopA or CopI were formed by incubation at 90 °C for 2 min followed by slow cooling to 37 °C in TMN buffer. Reaction mixtures (10 µl) contained end-labeled RNA (20,000 cpm) either free or in complex, carrier tRNA (2.5 µg), and lead(II) acetate at a final concentration of 8 or 16 mM in TMN buffer. Incubation was done at 37 °C for 5 min, and the reactions were stopped by the addition of 5 µl of 0.1 M EDTA followed by ethanol precipitation. Pellets were dissolved in 10 µl of stop buffer (93% formamide, 30 mM EDTA, 0.05% xylene cyanol, 0.05% bromphenol blue, 0.5% SDS). Before electrophoresis on 15% polyacrylamide-urea 8 M gels, the samples were heated at 90 °C for 3 min.

RNase III Hydrolysis

5'-End-labeled CopT was incubated with a 5-fold excess of CopA or CopI (10-7 M) in 10 µl of TMN buffer containing 1 mM dithiothreitol at 37 °C for 5, 15, or 30 min. Full RNA duplexes were formed as described above. Cleavage of free or complexed CopT was at 37 °C from 25 s to 10 min with 0.002 µg of RNase III or from 1 to 10 min with 0.25 µg of RNase III (a gift from D. Court). All reactions contained 10 µg of carrier tRNA. Reactions were stopped by phenol extraction followed by RNA precipitation. Pellets were dissolved in 10 µl of stop buffer and analyzed as described above.


RESULTS

Pb(II) Probing Distinguishes a Complete Duplex from a Kissing Complex

The primary binding intermediate involving the loop-loop interaction was studied by using the truncated antisense RNA, CopI, which is unable to promote a full duplex with CopT (6). In both free RNAs, the major cleavages occur in single-stranded regions, mainly in the hairpin loops and the interhelical regions (Fig. 1). The cleavage positions are shown schematically on the secondary structures of the RNAs (Fig. 2). The cleavages in loop II of CopI (but also CopA) and in loop II' of CopT extend into the helical regions, which is probably due to the presence of several bulged nucleotides (15). Under native conditions (kissing complex), CopI binding induces significant changes in the Pb(II) cleavage pattern of CopT (Fig. 1A, lanes 5 and 6). Strong protection is observed on the 3' side of the upper stem II' at U117 to A123 and to a lesser extent at U102 to C107. Furthermore, increased reactivity induced by CopI binding occurs at U109 and G112. The Pb(II) sensitivity of the non-complementary regions is not altered, indicating that CopI binding induces structural changes restricted to the upper part of stem loop II' of CopT (Fig. 1A). When full duplexes are formed, complete resistance to Pb(II) hydrolysis is observed in the region encompassing U94 to C138 (Fig. 1A, lanes 8 and 9). Therefore, Pb(II) is a sensitive probe that allows discrimination between a full duplex and the kissing complex. Conversely, binding of CopT induces enhanced Pb(II) cleavages at A29 and, to a lesser extent, at C28, A30, and A31 in CopI (Fig. 1A). Most of the nucleotides located in loop II of CopI are still cleaved by Pb(II) upon CopT binding, indicating that the upper part of helix II participates in the formation of the intermolecular helix that characterizes the kissing complex. Significant protection on both sides of the upper helix of CopI is observed, especially on the 5' side of helix II at U33 to A39 (Fig. 1B).


Fig. 1. Pb(II) probing and effect of binding of antisense RNA or CopT. A, Pb(II) hydrolysis on 5'-end-labeled CopT alone (lanes 1-3) or in an excess of truncated antisense CopI (lanes 4-9) or CopA (lanes 10-15). Complex formation was performed at 37 °C for 15 min (lanes 4-6 and 10-12) or after a denaturation-annealing treatment (lanes 7-9 and 13-15). Lanes 1, 4, 7, 10, and 13, incubation controls; lanes 2, 5, 8, 11, and 14, 8 mM Pb(II); lanes 3, 6, 9, 12, and 15, 16 mM Pb(II). B and C, Pb(II) hydrolysis on 5'-end-labeled CopI (B) or 5'-end-labeled CopA (C), alone or in the presence of an excess of CopT. Lanes 1, 2, and 2', incubation controls on free RNA (lane 1) or complexed with CopT (lanes 2 and 2'); lanes 3-5, 8 mM Pb(II); lanes 6 and 7, 16 mM Pb(II); lanes 4 and 7, free 5'-end-labeled RNA; complex formation was performed at 37 °C for 5 min (lanes 4 and 7) or 15 min (lanes 5 and 8); lanes T and L, RNase T1 and alkaline ladders, respectively.
[View Larger Version of this Image (65K GIF file)]


Fig. 2. Pb(II) probing and effect of binding of antisense RNA or CopT. The results are summarized on the secondary structure of CopA (left), CopI, and CopT (right). Positions of Pb(II) cleavages in the free RNAs are indicated by arrows. Large filled arrows, strong; small shaded arrows, moderate; and open head arrows, weak cleavages. The effect of antisense RNA or CopT binding is indicated as follows: strong (bullet ) and moderate (open circle ) protection. Enhancements are represented by stars proportional to the intensity. The labeled RNA used is indicated by an asterisk. The effects of CopT binding on CopI or CopI binding on CopT are shown in the insets, using the same symbols.
[View Larger Version of this Image (34K GIF file)]

Pb(II) Probing Shows That a Full Duplex Is Not the Major Product of CopA-CopT Binding

A CopA-CopT complex in which either CopA or CopT was labeled at the 5' end was formed under native conditions at 37 °C and subjected to Pb(II) probing. Under these conditions, stable complexes were formed within 5 min at 37 °C (data not shown). Unexpectedly, CopA binding induces the same reactivity changes in stem loop II' of CopT as CopI (cf. lanes 5 and 6 with lanes 11 and 12 in Fig. 1A). In particular, the bulged cytosine 101 and part of the external loop II' of CopT remain accessible to Pb(II). Additional protections occur in the 140-162 region (Fig. 2). No major structural rearrangement of CopT RNA occurs in the non-complementary region since the Pb(II) sensitivity remained unchanged (Fig. 1A). In contrast, the formation of a full CopA-CopT duplex entails complete protection of the CopA binding site from nucleotide 81 to 162 of CopT (Fig. 1A, lanes 14 and 15). Conversely, CopT binding induces the same reactivity changes in stem loop II of CopI and CopA (see Fig. 1, B and C). This is clearly illustrated by a strong enhancement at A60 and weaker increase at C59, A61, and A62 in CopA. In addition, the 5'-proximal 30 nucleotides of CopA are protected from Pb(II) in the presence of CopT (Fig. 2).

Altogether, these results suggest that a full duplex is not the major product of the CopA-CopT complex in vitro. The extended loop-loop contact, as found in the CopI-CopT complex, persists in the CopA-CopT complex, but this complex is additionally stabilized by the formation of an intermolecular helix that involves the 5'-proximal 30 nucleotides of CopA and the complementary region of CopT.

RNase III Cleavages in the CopA-CopT Complex Show That the Kissing Complex Is Slowly Converted to an Extended Duplex

RNase III was shown to process the CopA-CopT complex in vivo (9). Therefore, we reinvestigated RNase III-dependent cleavages under conditions of full duplex formation and native CopA-CopT complex formation to define the extent of duplex formation. The cleavage positions were mapped using 5'-end-labeled CopT RNA (Fig. 3A). The cleavage sites are indicated on a schematic drawing (Fig. 3B). When CopA or CopI was heated and slowly cooled in the presence of labeled CopT, allowing the formation of full RNA duplexes, the same concentration of RNase III was able to fully digest both duplexes. The strongest cleavages were located at A93, U94, U99, and U100 (Fig. 3A). Conversely, with the native CopA-CopT complex, RNase III produced two major cleavages in CopT at U149 and C154 (Fig. 3A). This result is in agreement with the primary RNase III cleavage identified at C154 in vivo and in vitro (14). However, weak cleavages corresponding to the presence of the full duplex were also found in CopT bound to CopA, especially after a prolonged time of incubation (at least up to 15 min, Fig. 1A). Under the same conditions, the CopI-CopT complex formed at 37 °C was completely resistant to RNase III hydrolysis (data not shown). Therefore, these experiments indicate that the full duplex is not the major product of CopA-CopT complex in vitro and that its formation appears to be a slow process in vitro.


Fig. 3. Analysis of RNase III cleavage positions in CopA-CopT complexes formed in vitro. A, RNase III hydrolysis of CopA-CopT complexes formed with 5'-end-labeled CopT. Native CopA-CopT complexes were formed at 37 °C for 5, 15, and 30 min, and full duplexes (FD) were obtained after a denaturation-annealing treatment. Reactions were done with 0.002 µg of RNase III (+) at 37 °C for 0.5 min (lane 1), 1 min (lane 2), 2 min (lane 3), and 5 min (lane 4). Free CopT (CopT*) was incubated in the absence (-) or in the presence of RNase III (+). Lanes T and U, RNase T1 and RNase U2 under denaturing conditions, respectively; lane L, alkaline ladder. The positions of the major RNase III cleavages are given. B, the positions of RNase III cleavages in CopT RNA bound to CopA under native conditions (top) or after a denaturation-annealing treatment (bottom) are shown. RNase III cuts are indicated by filled, shaded, and open arrows for strong, moderate, and weak cleavages, respectively. The Shine-Dalgarno sequence (SD) of tap is framed.
[View Larger Version of this Image (27K GIF file)]


DISCUSSION

Previous studies suggested that the binding of CopA to CopT proceeds in at least two experimentally distinguishable steps: kissing and hybridization duplex formation (5-7). The first, rate-limiting step is the formation of a transient loop-loop interaction (a so-called kissing complex) that was supposed to permit the subsequent formation of a complete RNA duplex. The present structural probing performed on the kissing complex shows that most of the nucleotides in loop II of the truncated antisense RNA CopI, as well as in the complementary loop, are accessible to Pb(II). However, since point mutations within the CGCC sequence in loop II of CopA strongly affect the pairing rate and result in copy number-up phenotypes (5, 11, 17), these nucleotides are inferred to be part of the primary kissing complex (Fig. 4B). This intermediate, not detectable by the present probing approach, rapidly rearranges to an extended kissing complex in which the 5' side of the upper part of stem II interacts in an interstrand pairing with CopT (Fig. 4C). This is further supported by RNase V1 cuts in the upper stem II of CopI and increased accessibility of the 3' side of loop II toward RNase T2 (6). Notably, the formation of the extended kissing complex induces a strong Pb(II) cleavage in the loop II of the antisense RNA. This cleavage may be due to the presence of a magnesium binding site. In line with this view, Gregorian and Crothers (18) showed that the uptake of at least two magnesium ions stabilized the loop-loop interaction in the RNAI-RNAII stem loop complex of plasmid ColE1.


Fig. 4. The binding pathway of CopA and CopT. Free CopA and CopT (A) interact with subsets of bases within their hexanucleotide loop sequences to form a reversible intermediate (B). By a rearrangement, a more stable and asymmetric extended kissing complex is formed (C) that facilitates hydrogen bonding of accessible single-stranded regions to yield the CopA-CopT complex (D). This long lived intermediate is converted very slowly to a full duplex (E).
[View Larger Version of this Image (18K GIF file)]

The Pb(II) probing experiment also demonstrates that the extended kissing complex persists in the CopA-CopT complex. Furthermore, extensive protection in the 5' segment of CopA and the complementary region of CopT supports the formation of 30-34 interstrand base pairs. We also showed that RNase III, which processes the CopA-CopT complex in vivo (9), cleaves the native CopA-CopT complex and the full duplex, respectively, in completely different ways. The major RNase III cleavage site in CopT bound to CopA under native conditions is centered within the 30-base pair intermolecular helix (Fig. 3B), which is consistent with known site preferences of RNase III (19). The extended kissing complex CopT-CopI that is not processed in vivo (9) is also resistant to RNase III hydrolysis in vitro. The RNase III experiments indicate that only a minor fraction of the CopA-CopT complex proceeds to a full duplex in vitro. This suggests that the asymmetric extended kissing complex induces a structural bend that brings together the two complementary single-stranded segments neighboring the hairpins II and II' to facilitate their rapid pairing (Fig. 4D). Since previous studies showed that stable CopA-CopT complexes were formed very rapidly (5), the identification of a stable and not fully base-paired CopA-CopT complex is somewhat unexpected. The present work indicates that this long lived intermediate (Fig. 4D) predominates for at least 15 min under conditions in which kissing complex formation occurs within a few seconds and that conversion to a full duplex is very slow, as it was proposed in the ColE1 system (4).

Similar conclusions for the binding pathway of the antisense RNA involved in translational control of repA in IncB plasmids have been reported (10). The antisense RNAI primarily inhibits the formation of a pseudoknot structure that activates repA translation (20). Both CopA and RNAI contain stable hairpins with identical hexanucleotide sequences within the loops. However, RNAI has only a short 5' tail (9 nucleotides) and lacks a 5' stem loop structure. Enzymatic probing indicated that RNAI and the target RNAII of plasmid pMU720 do not rapidly form a full duplex in vitro but rather form an extended kissing complex stabilized by additional base pairs involving the 5' tail of RNAI. In ColE1, NMR spectroscopy experiments showed that a kissing complex between two complementary stem loops of RNAI-RNAII involves base pairing of all loop residues (21). In this system, the stems of both hairpins are rather short (6 base pairs) and lack bulges. Comparing the different structures of the RNAI-RNAII stem loop complex (ColE1) with that of CopI (or CopA)-CopT (R1) and RNAI-RNAII (pMU720), it is tempting to speculate that bulges near a recognition loop permit an induced rearrangement to obtain a transient but stable extended kissing complex. Removal of the bulged residues in stem II of CopA and stem II' of CopT strongly decreases both binding rate and kissing complex stability (22).

The same major RNase III cleavage site was identified in vitro and in vivo at C154 in CopT RNA (9). Thus, the majority of the CopA-CopT complexes that inhibit repA expression in vivo may not be fully base-paired. The extent of complete pairing in vivo may be underestimated due to RNase III processing followed by very rapid decay of the RNAs. Nevertheless, we propose that the formation of the long lived CopA-CopT complex detected in vitro is sufficient to account for the observed translational control. We have recently shown that this complex prevents initiation complex formation at the tap ribosome binding site (23). Even the extended kissing complex (CopI-CopT) was able to transiently interfere with ribosome binding that correlates well with in vivo results (9). In agreement with previous observations (24), the present data also show that the binding of CopI or CopA does not induce major conformational changes in the non-complementary sequences of CopT, i.e. in the tap and repA ribosome binding site regions. That Pb(II) is very sensitive to subtle conformational changes suggests that the structure of the kissing complex itself, rather than an induced RNA structure change downstream, prevents ribosome binding at tap. In the wild type situation, the presence of the 5' segment of CopA stabilizes the transient kissing complex, providing approximately three helical turns of double-stranded RNA immediately 5' of the tap Shine-Dalgarno sequence, which is sufficient to completely and irreversibly block tap translation.

The finding that complete pairing between an antisense and a target RNA is slow and can be unnecessary for inhibition may illustrate a rule rather than an exception. Structurally complex recognition stem loops of antisense and target RNAs appear to be intrinsically difficult to unfold during complex formation, and hence many biological control systems may have evolved to employ binding intermediates active in the inhibitory step. An advantage of such a mechanism is that inhibition kinetics can be faster than pairing kinetics (e.g. see Ref. 25) since the involved stem loops appear optimized for binding rate (26, 27), whereas unfolding rates may be difficult to optimize (unless unwinding activities are present). We suggest that such considerations may be relevant for the design of efficient antisense RNA inhibitors.


FOOTNOTES

*   This work was supported by grants from CNRS, Ministère des Affaires Étrangères (MAE) through a Program International de Coopération Scientifique (PICS) (to P. R.), and by the Swedish Natural Science Research Council (NFR) and Swedish Research Council for Engineering Sciences (TFR) (to G. W.).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.
Dagger    Supported by a fellowship from the Swedish Institute.
par    To whom correspondence should be addressed. Tel.: 33-3-88-41-70-51; Fax: 33-3-88-60-22-18; E-mail: romby{at}ibmc.U-strasbg.fr.

ACKNOWLEDGEMENTS

We thank Eric Westhof and Hervé Moine for stimulating discussions and Steve Lodmell for critical reading of the manuscript.


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