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.
Kolb
F A
FA
UPR 9002 du Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France.
Malmgren
C
C
Westhof
E
E
Ehresmann
C
C
Ehresmann
B
B
Wagner
E G
EG
Romby
P
P
D001426:Bacterial Proteins / Q000378:metabolism*; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent; D003198:Computer Simulation; D019216:Metals, Heavy / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020871:RNA Stability; D016372:RNA, Antisense / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020038:RNA, Spliced Leader / Q000378:metabolism
D001426:Bacterial Proteins; D002413:Cations, Divalent; C071263:CopA protein, Bacteria; C073592:CopT protein, Plasmid R1; D019216:Metals, Heavy; D016372:RNA, Antisense; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D020038:RNA, Spliced Leader