Bulged residues promote the progression of a loop-loop interaction to a stable and inhibitory antisense-target RNA complex.

In several groups of bacterial plasmids, antisense RNAs regulate copy number through inhibition of replication initiator protein synthesis. These RNAs are characterized by a long hairpin structure interrupted by several unpaired residues or bulged loops. In plasmid R1, the inhibitory complex between the antisense RNA (CopA) and its target mRNA (CopT) is characterized by a four-way junction structure and a side-by-side helical alignment. This topology facilitates the formation of a stabilizer intermolecular helix between distal regions of both RNAs, essential for in vivo control. The bulged residues in CopA/CopT were shown to be required for high in vitro binding rate and in vivo activity. This study addresses the question of why removal of bulged nucleotides blocks stable complex formation. Structure mapping, modification interference, and molecular modeling of bulged-less mutant CopA-CopT complexes suggests that, subsequent to loop-loop contact, helix propagation is prevented. Instead, a fully base paired loop-loop interaction is formed, inducing a continuous stacking of three helices. Consequently, the stabilizer helix cannot be formed, and stable complex formation is blocked. In contrast to the four-way junction topology, the loop-loop interaction alone failed to prevent ribosome binding at its loading site and, thus, inhibition of RepA translation was alleviated.

Four-way junctions in antisense RNA-mRNA complexes involved in plasmid replication control: a common theme?

In several groups of bacterial plasmids, antisense RNAs regulate copy number through inhibition of replication initiator protein synthesis. In plasmid R1, we have recently shown that the inhibitory complex between the antisense RNA (CopA) and its target mRNA (CopT) is characterized by the formation of two intermolecular helices, resulting in a four-way junction structure and a side-by-side helical alignment. Based on lead-induced cleavage and ribonuclease (RNase) V(1) probing combined with molecular modeling, a strikingly similar topology is supported for the complex formed between the antisense RNA (Inc) and mRNA (RepZ) of plasmid Col1b-P9. In particular, the position of the four-way junction and the location of divalent ion-binding site(s) indicate that the structural features of these two complexes are essentially the same in spite of sequence differences. Comparisons of several target and antisense RNAs in other plasmids further indicate that similar binding pathways are used to form the inhibitory antisense-target RNA complexes. Thus, in all these systems, the structural features of both antisense and target RNAs determine the topologically possible and kinetically favored pathway that is essential for efficient in vivo control.

Progression of a loop-loop complex to a four-way junction is crucial for the activity of a regulatory antisense RNA.

The antisense RNA, CopA, regulates the replication frequency of plasmid R1 through inhibition of RepA translation by rapid and specific binding to its target RNA (CopT). The stable CopA-CopT complex is characterized by a four-way junction structure and a side-by-side alignment of two long intramolecular helices. The significance of this structure for binding in vitro and control in vivo was tested by mutations in both CopA and CopT. High rates of stable complex formation in vitro and efficient inhibition in vivo required initial loop-loop complexes to be rapidly converted to extended interactions. These interactions involve asymmetric helix progression and melting of the upper stems of both RNAs to promote the formation of two intermolecular helices. Data presented here delineate the boundaries of these helices and emphasize the need for unimpeded helix propagation. This process is directional, i.e. one of the two intermolecular helices (B) must form first to allow formation of the other (B'). A binding pathway, characterized by a hierarchy of intermediates leading to an irreversible and inhibitory RNA-RNA complex, is proposed.

Probing the structure of RNAIII, the Staphylococcus aureus agr regulatory RNA, and identification of the RNA domain involved in repression of protein A expression.

RNAIII, a 514-nt RNA molecule, regulates the expression of many Staphylococcus aureus genes encoding exoproteins and cell-wall-associated proteins. We have studied the structure of RNAIII in solution, using a combination of chemical and enzymatic probes. A model of the secondary structure was derived from experimental data with the help of computer simulation of RNA folding. The model contains 14 hairpin structures connected by unpaired nucleotides. The data also point to three helices formed by distant nucleotides that close off structural domains. This model was generally compatible with the results of in vivo probing experiments with dimethylsulfate in late exponential-phase cultures. Toe-printing experiments revealed that the ribosome binding site of hld, which is encoded by RNAIII, was accessible to the Escherichia coli 30S ribosomal subunit, suggesting that the in vitro structure represented a translatable form of RNAIII. We also found that, within the 3' end of RNAIII, the conserved hairpin 13 and the terminator form an intrinsic structural domain that exerts specific regulatory activity on protein A gene expression.

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 antisense RNA CopA binds to the leader region of the repA mRNA (target: CopT). Previous studies on CopA-CopT pairing in vitro showed that the dominant product of antisense RNA-mRNA binding is not a full RNA duplex. We have studied here the structure of CopA-CopT complex, combining chemical and enzymatic probing and computer graphic modeling. CopI, a truncated derivative of CopA unable to bind CopT stably, was also analyzed. We show here that after initial loop-loop interaction (kissing), helix propagation resulted in an extended kissing complex that involves the formation of two intermolecular helices. By introducing mutations (base-pair inversions) into the upper stem regions of CopA and CopT, the boundaries of the two newly formed intermolecular helices were delimited. The resulting extended kissing complex represents a new type of four-way junction structure that adopts an asymmetrical X-shaped conformation formed by two helical domains, each one generated by coaxial stacking of two helices. This structure motif induces a side-by-side alignment of two long intramolecular helices that, in turn, facilitates the formation of an additional intermolecular helix that greatly stabilizes the inhibitory CopA-CopT RNA complex. This stabilizer helix cannot form in CopI-CopT complexes due to absence of the sequences involved. The functional significance of the three-dimensional models of the extended kissing complex (CopI-CopT) and the stable complex (CopA-CopT) are discussed.