Antisense RNA-mediated transcriptional attenuation in plasmid pIP501: the simultaneous interaction between two complementary loop pairs is required for efficient inhibition by the antisense RNA
Streptococcal plasmid pIP501 uses antisense RNA-mediated transcriptional attenuation to regulate its replication. Previous in vitro assays suggested that binding intermediates between RNAII (sense RNA) and RNAIII (antisense RNA) are sufficient for inhibition, and a U-turn structure on RNAII loop L1 was found to be crucial for the interaction with RNAIII. Here, sequence and structural requirements for an efficient RNAII–RNAIII interaction were investigated. A detailed probing of RNA secondary structure combined with in vitro single-round transcription assays indicated that complex formation between the two molecules progresses into the lower stems of both loop pairs of the sense and antisense RNAs, but that the complex between RNAII and RNAIII is not a full duplex. Stem–loops L3 and L4 were required to be linked to one other for efficient contact with the complementary loops L2 and L1 of the sense RNA, indicating a simultaneous interaction between these two loop pairs. Thereby, the sequence and length of the spacer connecting L3 and L4 were shown not to be important for inhibition.
AsanoK.,
MizobuchiK.2000; Structural analysis of late intermediate complex formed between plasmid ColIb-P9 Inc RNA and its target RNA. How does a single antisense RNA repress translation of two genes at different rates?. J Biol Chem 275:1269–1274[CrossRef]
BrantlS.1994; The copR gene product of plasmid pIP501 acts as a transcriptional repressor at the essential repR promoter. Mol Microbiol 14:473–483[CrossRef]
BrantlS.2004; Plasmid replication controlled by antisense RNAs. In The Biology of Plasmids, chapter 3 pp 47–62 Edited by
FunnellB.,
PhillipsG.
Washington, DC: AMS Press;
BrantlS.,
WagnerE. G. H.1994; Antisense RNA-mediated transcriptional attenuation occurs faster than stable antisense/target RNA pairing: an in vitro study of plasmid pIP501. EMBO J 13:3599–3607
BrantlS.,
WagnerE. G. H.1996; An unusually long-lived antisense RNA in plasmid copy number control: in vivo RNAs encoded by the streptococcal plasmid pIP501. J Mol Biol 255:275–288[CrossRef]
BrantlS., Birch-HirschfeldE.,
BehnkeD.1993; RepR protein expression on plasmid pIP501 is controlled by an antisense RNA-mediated transcription attenuation mechanism. J Bacteriol 175:4052–4061
GreenfieldT. J., FranchT., GerdesK.,
WeaverK. E.2001; Antisense RNA regulation of the par post-segregational killing system: structural analysis and mechanism of binding of the antisense RNA, RNAII and its target, RNAI. Mol Microbiol 42:527–537[CrossRef]
GubbinsM. J., ArthurD. C., GhetuA. F., GloverJ. N. M.,
FrostL. S.2003; Characterizing the structural features of RNA/RNA interactions of the F-plasmid FinOP fertility inhibition system. J Biol Chem 278:27663–27671[CrossRef]
HeidrichN.,
BrantlS.2003; Antisense-RNA mediated transcriptional attenuation: importance of a U-turn loop structure in the target RNA of plasmid pIP501 for efficient inhibition by the antisense RNA. J Mol Biol 333:917–929[CrossRef]
HjaltT.,
WagnerE. G. H.1992; The effect of loop size in antisense and target RNAs on the efficiency of antisense RNA control. Nucleic Acids Res 20:6723–6732[CrossRef]
HjaltT.,
WagnerE. G. H.1995; Bulged-out nucleotides in an antisense RNA are required for rapid target RNA binding in vitro and inhibition in vivo . Nucleic Acids Res 23:580–587[CrossRef]
KolbF. A., EngdahlH. M., EhresmannB., EhresmannC., WesthofE., WagnerE. G. H.,
RombyP., Slagter-JägerJ. G.2000a; Progression of a loop-loop complex to a four-way junction is crucial for the activity of a regulatory antisense RNA. EMBO J 19:5905–5915[CrossRef]
KolbF. A., MalmgrenC., WesthofE., EhresmannC., EhresmannB., WagnerE. G. H.,
RombyP.2000b; 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. RNA 6:311–324[CrossRef]
KolbF. A., WesthofE., EhresmannB., EhresmannC., WagnerE. G. H.,
RombyP.2001; Four-way junctions in antisense RNA-mRNA complexes involved in plasmid replication control: a common theme?. J Mol Biol 309:605–614[CrossRef]
MalmgrenC., WagnerE. G. H., EhresmannC., EhresmannB.,
RombyP.1997; Antisense RNA control of plasmid R1 replication: the dominant product of the antisense RNA-mRNA binding is not a full RNA duplex. J Biol Chem 272:12508–12512[CrossRef]
RasmussenA. A., EriksenM., GilanyK., UdesenC., FranchT., PetersenC.,
Valentin-HansenP.2005; Regulation of ompA mRNA stability: the role of a small regulatory RNA in growth phase-dependent control. Mol Microbiol 58:1421–1429[CrossRef]
ThistedT., SørensenN., WagnerE. G. H.,
GerdesK.1994; Mechanism of postsegregational killing: Sok antisense RNA interacts with Hok mRNA via its 5′-end single-stranded leader and competes with the 3′-end of Hok mRNA for binding to the mok translational initiation region. EMBO J 13:1960–1968
UdekwuK. I., DarfeuilleF., VogelJ., ReimegardJ., HolmqvistE.,
WagnerE. G. H.2005; Hfq-dependent regulation of OmpA synthesis is mediated by an antisense RNA. Genes Dev 19:2355–2366[CrossRef]
WagnerE. G. H., AltuviaS.,
RombyP.2002; Antisense RNAs in bacteria and their genetic elements. In Adv in Genetics pp 361–398 Edited by
DunlapJ. C.,
WuC.
London: Academic Press;
Antisense RNA-mediated transcriptional attenuation in plasmid pIP501: the simultaneous interaction between two complementary loop pairs is required for efficient inhibition by the antisense RNA