Mechanism of binding of the antisense and target RNAs involved in the regulation of IncB plasmid replication

Abstract

The replication frequency of the IncB miniplasmid pMU720 is dependent upon the expression of the repA gene. Binding of a small, highly structured, antisense RNA (RNA I) to its complementary target in the RepA mRNA (RNA II) inhibits repA expression and thus regulates replication. Analyses of binding of RNA I to RNA II indicated that the reaction consists of three major steps. The first step, initial kissing complex formation, involves base pairing between complementary sequences in the hairpin loops of RNA I and RNA II. The second step is facilitated by interior loop structures in the upper stems of RNA I and RNA II and involves intrastrand melting and interstrand pairing of the upper stem regions to form an extended kissing complex. This complex was shown to be sufficient for inhibition ofrepA expression. The third step involves stabilization of the extended kissing complex by pairing between complementary single-stranded tail regions of RNA I and RNA II. Thus, the final product of RNA I-RNA II binding is not a full duplex between the two molecules.

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

“…The overall topology structure of the extended kissing complex is similar to some other RNA four-way junctions (Krol et al+, 1990;Walter et al+, 1998a;Nowakowski et al+, 1999) and DNA Holliday junctions (Duckett et al+, 1992)+ In both DNA and RNA four-way junctions, divalent ions are required for the formation and stabilization of antiparallel X-shaped structures (Duckett et al+, 1992;Walter et al+, 1998b)+ The particularity of the proposed CopA-CopT structure is the crossing over of the strands at the junction under the constraints imposed by the two loops connecting intermolecular helices B and B9+ This forces a side-by-side alignment of the two helical domains that brings the 59 tail of CopA in close proximity to the complementary region of CopT (Fig+ 7)+ The formation of intermolecular helix C, which clamps the two long helical domains, greatly enhances the stability of the complex (Persson et al+, 1990a;Malmgren et al+, 1997)+ Crystallographic analysis of a group I ribozyme domain revealed a similar organization (Cate et al+, 1996): a sharp bend induced by an internal loop allows a side-by-side alignment of two helical domains that is additionally stabilized by metal-and ribosemediated backbone contacts and two long-range tertiary interactions+ A side-by-side configuration was also proposed for the hairpin ribozyme, here stabilized by interactions between two internal loops (Earnshaw et al+, 1997)+ The formation of a stable RNA-RNA complex is not unique to CopA-CopT, and is also a key feature in the replication control of plasmids belonging to the IncB and IncIa groups (Siemering et al+, 1994; plasmids+ In these systems, the antisense RNAs inhibit the formation of a pseudoknot structure that activates rep translation (Wilson et al+, 1993;)+ All these antisense and target RNAs are characterized by stable hairpins with identical loop sequences and bulged residues in the upper stem regions+ Enzymatic probing performed on (antisense) RNAI in pMU720 plasmid bound to its target indicated that a full duplex was not rapidly formed in vitro+ Instead, binding resulted in an extended kissing complex stabilized by 59 tail interactions (Siemering et al+, 1994)+ One may therefore speculate that, in all these systems, the final product of the binding reaction in vitro is characterized by an overall topology very similar to that reported here, except that the lengths of helices B and B9, if formed in the IncB/IncIa cases, could be different+…”

“…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+…”

“…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+…”

Antisense-RNA-mediated plasmid copy number control in pCG1-family plasmids, pCGR2 and pCG1, in Corynebacterium glutamicum

“…Antisense RNAs negatively regulating replication of bacterial plasmids are typically small (50-250 bp), short-lived, constitutively synthesized, cis-encoded, single-stranded RNA molecules that are complementary to the 59 leader region of target mRNAs (Brantl, 2002;Khan, 1997;Rasooly & Rasooly, 1997). The currently known antisense-RNA-mediated control mechanisms include transcription attenuation (Heidrich & Brantl, 2007;Le Chatelier et al, 1996;Novick et al, 1989), translation inhibition (del Solar & Espinosa, 2000), inhibition of primer maturation (Davison, 1984) and inhibition of pseudoknot formation (Athanasopoulos et al, 1999;Siemering et al, 1994). Antisense RNAs and their target mRNAs are generally highly structured, containing stemloop(s).…”