Control of replication of plasmid R1: formation of an initial transient complex is rate-limiting for antisense RNA-target RNA pairing.

Abstract

The replication frequency of plasmid R1 is determined by the availability of the initiator protein RepA. Synthesis of RepA is negatively controlled by an antisense RNA, CopA, which forms a duplex with the upstream region of the RepA mRNA, CopT. We have previously shown that the in vitro formation of the CopA‐CopT duplex follows second‐order kinetics and occurs in at least two steps. The first step is the formation of a transient (kissing) complex, which is subsequently converted to a persistent duplex. Here, we investigate the details of the reaction scheme and determine the rate constants of the pathway from the free RNAs to the complete duplex. Using a shortened CopA RNA (CopI) we have been able to determine the association and dissociation rate constants (k1,k‐1) for the kissing complex (which are inferred to be the same for CopI‐T and CopA‐T), and measured the hybridization rate constant k2 (for CopA‐T k2 is at least 1000‐fold greater than for CopI‐T). The analysis of CopA derivatives of mutant and wild‐type origin shows that the rate of formation of the kissing complex is rate‐limiting for the overall pairing reaction between CopA and CopT, both in vitro and in vivo. The biological implications of the kinetically irreversible RNA‐RNA binding reaction scheme are discussed.

RepR protein expression on plasmid pIP501 is controlled by an antisense RNA-mediated transcription attenuation mechanism

“…Interaction of such highly structured antisense RNA molecules with their targets is likely to involve several steps and starts with the formation of so-called kissing complexes between the singlestranded loops of antisense and target RNA. This concept was initially proposed by for the interaction of RNAI and RNAII of ColEl and was later supported by both in vivo and in vitro studies of the interaction of the CopA antisense RNA of plasmid R1 with its target CopT (31)(32)(33). In vitro experiments with the R1 system even suggested that formation of the kissing complex is sufficient for the inhibitory action of CopA to occur (43).…”

“…The topological organization of the replication region of pIP501 exhibits similarity to that of the gram-negative plasmid R1 (6,26). Synthesis of the RepA initiator protein of R1 is also controlled by an antisense RNA complementary to the leader region of the repA mRNA (26,32,33). It has, however, recently been found that the CopA antisense RNA of R1 acts at the translational level rather than on transcription.…”

An unusual structure formed by antisense-target RNA binding involves an extended kissing complex with a four-way junction and a side-by-side helical alignment

“…Previous kinetic and mutational analyses showed that binding is initiated via a loop-loop interaction between the 59-CGCC-39 sequence in the hairpin loop of CopA and the complementary sequence of CopT (Persson et al+, 1988(Persson et al+, , 1990b)+ However, these initial interactions are transient, as the present study of CopA-CopT and CopI-CopT complexes shows that most of the nucle- -H2), or in the presence of CopT-H2 (ϩ CopT-H2)+ Lanes U, L: RNase U2 and alkaline ladders, respectively+ C: Effect of magnesium concentration on Pb 2ϩ hydrolysis+ The intensity of the band corresponding to cleavage at U47 of CopI-H2 complexed to CopT-H2 is depicted as a function of magnesium concentration+ otides in loop II of the antisense RNA, as well as in the complementary target loop, are accessible to singlestrand-specific probes+ Thus, helix extension from this initial loop-loop complex generates helices B and B9, resulting in disruption of the initiating base pairs and formation of the extended kissing complex (Fig+ 6C)+ The enzymatic and chemical probing data indicate that nucleotides on both sides of the upper stem II segments (48-56, 63-68) of CopA and CopI interact by interstrand pairing with the complementary nucleotides in CopT forming the two intermolecular helices B and B9 (Fig+ 6C)+ Further support comes from the observation that mutations in helices B/B9 that disrupt the complementarity between CopA or CopI and CopT affect the structure of the extended kissing complex+ On the other hand, mutations below the lower bulge had no effect on the formation of helices B and B9+ Taken together, these results define the boundaries of helices B and B9 (Fig+ 6C) and indicate that progression of the intermolecular helices B and B9 is arrested due to topological stress+ Consequently, the intramolecular helices A and A9 remain identical in CopI-CopT and CopACopT complexes+ We infer that conversion of the initial loop-loop interaction to the four-helix junction structure is facilitated by low stability of the upper stem regions, conferred by the bulged residues and the presence of several G-U and A-U base pairs in both RNAs+ By independent experiments, it was previously shown that removal of bulges severely impaired binding rates and in vivo control (Hjalt & Wagner, 1995)+ The results presented here also indicate that complete pairing between CopA and CopT does not occur rapidly in vitro+ It is likely that the formation of the peculiar four-helix junction structure results in kinetic or topological entrapment of the complex for extended periods of time (Malmgren et al+, 1997; this work)+ The extended kissing complex adopts an X-shaped structure with a side-by-side alignment of helical domains Molecular modeling was used to deduce a global fold of the stable CopA-CopT complex+ The validity of the overall architecture presented relies on a variety of experimental data (chemical and enzymatic probing; effect of mutations on CopA-CopT structure) that were used to assemble a structure with appropriate stereochemistry+ We propose that the folding of the extended kissing complex at the four-way junction adopts an asymmetric stacked cruciform structure, formed by the colinear helices B-A and B9-A9 in a parallel configuration (Figs+ 6C and 7)+ This model predicts a close contact between helices at the stacked cruciform junction (Fig+ 7)+ Such a negatively charged cavity is likely to bind divalent ions with high affinity+ Interestingly, computer modeling based on Brownian-dynamics simulations (Hermann & Westhof, 1998) predicted a magnesium-binding site coordinated via the phosphate groups betwee...…”

“…CopA binds to the leader region of the repA mRNA (CopT), located about 80 nt upstream of the repA start codon (Fig+ 1)+ Binding prevents tap translation and thereby repA expression (Blomberg et al+, , 1994Malmgren et al+, 1996)+ The CopA-CopT binding process is viewed as a series of reactions leading to progressively more stable complexes (Persson et al+, 1988(Persson et al+, , 1990a(Persson et al+, , 1990bMalmgren et al+, 1997)+ CopA and CopT are fully complementary and both RNAs contain a major stem-loop structure (II/II9 in Fig+ 1) that is essential for high pairing rates and control (Öhman & Wagner, 1989;Hjalt & Wagner, 1992, 1995+ The initial step involves a transient looploop interaction (kissing complex) between the complementary hairpin loops (Persson et al+, 1990a(Persson et al+, , 1990b)+ Indeed, a truncated CopA (CopI, Fig+ 1), lacking the 59 proximal 30 nt and consisting only of the major stemloop, does not form stable duplexes with CopT, but is capable of competing with CopA for binding (Persson et al+, 1990b)+ It was recently shown that in both CopICopT and CopA-CopT complexes, initial kissing is rapidly followed by more extended intermolecular interactions (Malmgren et al+, 1997)+ Subsequently, the single-stranded region in the 59 tail of CopA pairs with its complement in CopT to yield the stable, inhibitory CopA-CopT complex+ This complex is the dominant product of binding in vitro (Malmgren et al+, 1996(Malmgren et al+, , 1997+ Complete duplex formation is very slow and has been proposed to be irrelevant for control (Malmgren et al+, 1996(Malmgren et al+, , 1997Wagner & Brantl, 1998)+ Different pairing pathways that result in rapid formation of stable antisense-target RNA complexes have been described (Kittle et al+, 1989;Persson et al+, 1990b;Tomizawa, 1990;Siemering et al+, 1994;Thisted et al+, 1994)+ A common feature is the use of a restricted single-stranded region in each interacting RNA for the initial step+ In most cases, binding initiates between two loops, in some cases between a loop and a singlestranded RNA segment+ Subsequently, more stable complexes are either formed by the pairing of distal RNA segments or, in the latter case, by extension of the first helix+…”

“…In the plasmids of the ColE1-family, control of replication is mediated by an antisense RNA, RNAI, that interacts with the preprimer, RNAII, via initial and transient base pairing between complementary loops + NMR studies were performed on two RNA hairpins carrying sevenmembered complementary loops derived from RNAI/ RNAII (Marino et al+, 1995;Lee & Crothers, 1998)+ These studies indicated that all seven loop bases were paired in the loop-loop helix, and continuous stacking of the loop nucleotides on the 39 side of their respective stems was observed+ Loop-loop interactions in plasmid R1 (IncFII plasmid; Persson et al+, 1990aPersson et al+, , 1990bMalmgren et al+, 1997), pMU720 (IncB plasmid; Siemering et al+, 1994), and ColIb-P9 (IncIa plasmid; Asano et al+, 1998) FIGURE 1. Sequences and secondary structures of the antisense RNA (CopA) and of the leader segment of the repA mRNA+ Structures are based on chemical and enzymatic probing (Öhman & Wagner, 1989; this work)+ The target sequence (CopT) of the antisense RNA (CopA) is shown in brackets+ The Shine and Dalgarno (SD), stop codon of tap, and start codons of tap and repA are indicated+ Binding of CopA prevents translation of the tap reading frame by occluding ribosome binding at tap initiation site (Ϫ)+ Under these conditions, the stable RNA stem-loop that sequesters the repA ribosome binding site (RBS) prevents translation of the repA reading frame (Blomberg et al+, 1994)+ If CopA fails to bind, ribosomes translate the tap reading frame, terminate at the tap stop codon, and reinitiate at the repA RBS by a direct translational coupling (Blomberg et al+, , 1994)+ The sequence of the truncated antisense RNA (CopI) sufficient to inhibit tap translation is boxed Malmgren et al+, 1996)+ are clearly similar+ In all these systems, it was suggested that an initial loop-loop interaction is rapidly converted to an extended kissing complex+ This requires partial melting of the upper stem regions, most probably facilitated by the presence of bulged residues (Siemering et al+, 1994;Hjalt & Wagner, 1995)+ Interestingly, the extended kissing complex suffices for inhibition in vivo Wilson et al+, 1993)+ In the case of plasmid R1, the extended kissing complex is also capable of blocking ribosome binding at the tap translation initiation site in vitro (Malmgren et al+, 1996), suggesting the existence of a bulky structure+…”