Characterizing the structural features of RNA/RNA interactions of the F-plasmid FinOP fertility inhibition system.
F-like plasmid transfer is mediated by the FinOP fertility inhibition system. Expression of the F positive regulatory protein, TraJ, is controlled by the action of the antisense RNA, FinP, and the RNA-binding protein FinO. FinO binds to and protects FinP from degradation and promotes duplex formation between FinP and traJ mRNA, leading to repression of both traJ expression and conjugative F transfer. FinP antisense RNA secondary structure is composed of two stem-loops separated by a 4-base single-stranded spacer and flanked on each side by single-stranded tails. Here we show that disruption of the expected Watson-Crick base pairing between the loops of FinP stem-loop I and its cognate RNA binding partner, traJ mRNA stem-loop Ic, led to a moderate reduction in the rate of duplex formation in vitro. In vivo, alterations of the anti-ribosome binding site region in the loop of FinP stem-loop I reduced the ability of the mutant FinP to mediate fertility inhibition and to inhibit TraJ expression when expressed in trans at an elevated copy number. Alterations of intermolecular complementarity between the stems of these RNAs reduced the rate of duplex formation. Our results suggest that successful interaction between stem-loop I of FinP and stem-loop Ic of traJ mRNA requires that base pairing must proceed from an initial loop-loop interaction through the top portion of the stems for stable duplex formation to occur.
Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada.
Michael J
MJ
Gubbins
David C
DC
Arthur
Alexandru F
AF
Ghetu
J N Mark
JN
Glover
Laura S
LS
Frost
D001483:Base Sequence; D015152:Blotting, Northern; D003227:Conjugation, Genetic / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D005144:F Factor / Q000235:genetics*; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D016372:RNA, Antisense / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics*; D014158:Transcription, Genetic
D029968:Escherichia coli Proteins; D016372:RNA, Antisense; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; C074275:FinO protein, E coli; D012313:RNA