FinO is an RNA chaperone that facilitates sense-antisense RNA interactions

Abstract

The protein FinO represses F-plasmid conjugative transfer by facilitating interactions between the mRNA of the major F-plasmid transcriptional activator, TraJ, and an antisense RNA, FinP. FinO is known to bind stem-loop structures in both FinP and traJ RNAs; however, the mechanism by which FinO facilitates sense-antisense pairing is poorly understood. Here we show that FinO acts as an RNA chaperone to promote strand exchange and duplexing between minimal RNA targets derived from FinP. This strongly suggests that FinO may function to destabilize internal secondary structures within FinP and traJ RNAs that would otherwise act as a kinetic trap to sense-antisense pairing. The energy for FinO-catalyzed base-pair destabilization does not arise from ATP hydrolysis but appears to be supplied directly from FinO RNA binding free energy. An analysis of the activities of mutants that are specifically deficient in strand exchange but not RNA-binding activity demonstrates that strand exchange is essential to the ability of FinO to mediate sense-antisense RNA recognition, and that this function also plays a role in repression of conjugation in vivo.

Fig. 1. The structures of FinO and its RNA targets. (A) The sequence and secondary structure of RNA molecules used in this study. The 5′ untranslated region of traJ mRNA (which duplexes with FinP) is shown with the start codon (AUG) and ribosomal binding site (RBS) boxed. The A and B strands of SII and SIIΔ RNA duplexes are aligned to show the regions of base-pair complementarity. (B) Ribbons representation of FinO. The structure of FinO(26–186) (Ghetu et al., 2000) is shown, with a dotted line representing the unstructured N-terminal 25 residues. The Trp36 side-chain is displayed and the N-terminal residues 33–46 are highlighted in black. The amino acid sequence of the N-terminal region of FinO that is critical for strand exchange activity with the RNA duplex is shown above the structure. Finally, position 61 is shown on the structure in reference to the C-terminal truncation fragment FinO(1–61).

Fig. 2. FinO can catalyze strand exchange in duplex RNA substrates. (A) Schematic diagram of the RNA strand exchange assay. The SII RNA duplex is 5′-labeled with ³²P on the A strand (star) and is incubated with FinO and a molar excess of the unlabeled SII(A) strand at 37°C. Release of ³²P-labeled SII(A) strand is monitored by native gel electrophoresis over a 2 h time-course. (B) Comparison of strand exchange efficiencies between FinO and various N- and C-terminal truncated fragments. SII RNA was incubated with either FinO, FinO(26–186), FinO(45–186) or FinO(1–61) (each at a final concentration of 1 µM), or no protein. SIIΔ RNA was incubated with FinO at a final concentration of 1 µM. Aliquots were taken at 0, 1, 5, 15, 30, 60, 90 and 120 min after the start of the reaction and loaded directly onto a continuously running gel. (C) The percentage of ³²P-labeled SII(A) strand released from the duplex was plotted as a function of time and the apparent first order rate constant, k₁, was determined from this plot for FinO (filled circles), FinO(26–186) (filled triangles), no protein (filled squares) and FinO(1–61) (filled diamonds) (see Materials and methods). The percentage of ³²P-labeled SIIΔ(A) strand released from the SIIΔ duplex was similarly plotted as a function of time for FinO (open circles). The relative rates of strand exchange as a fraction of FinO, and their standard deviations (derived from at least three independent rate determinations) are shown. (D) Strand exchange of SII RNA after a 120-min reaction was measured as a function of protein concentration for FinO and the indicated mutants. Also shown is strand exchange of SIIΔ RNA after a 120-min incubation with FinO. The protein concentrations (in µM) are indicated at the top of each lane.

Fig. 3. Sequences near the N-terminus of FinO α1 are critical for RNA strand exchange Samples were incubated for 120 min with the indicated double (A and B) or single (C and D) alanine point mutants and strand exchange was detected by the release of ³²P-labeled SII(A) strand from the SII RNA duplex. The percentage of ³²P-labeled SII(A) strand released from duplex is presented in graphical form for the double (B) and single (D) alanine point mutants (standard deviations represent results from at least three independent experiments). As controls, strand exchange of SII RNA in the presence of FinO, FinO(45–186) or no protein was performed in parallel.

Fig. 4. FinO RNA strand exchange mutants are deficient in facilitating sense–antisense RNA interactions. (A) FinO, FinO(26–186) and FinO(45–186), each at a final concentration of 1 µM, and a no protein control were tested for their ability to facilitate sense–antisense pairing between SLII_x and SLIIc_x RNAs. Aliquots were taken at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4 and 10 min for FinO; 1, 2, 4, 8, 15, 30 and 60 min for FinO(26–186) and FinO(1–61); and 0, 10, 20, 30 and 60 min for FinO(45–186) and the no protein control. (B) Two-hour duplexing reactions were performed in the presence of FinO and the various FinO mutants indicated. (C) The amount of SLII_x duplexed at 2 h, expressed as a percentage of the amount of duplex generated for wild-type FinO, is displayed in graphical form. Error bars represent standard deviations calculated from at least three independent experiments.

Fig. 5. RNA strand exchange deficient FinO mutants bind RNA more tightly than wild-type FinO. Representative gel EMSAs for FinO, FinO(26–186) or FinO(45–186) binding to SLII RNA. Samples containing 50 pM SLII were incubated with protein at the concentrations indicated. These and similar experiments were used to determine the relative FinO–RNA association constants shown in Table I.

Fig. 6. In vivo stabilization of FinP by FinO and FinO derivatives. Stabilization of FinP in cells expressing FinP and the indicated FinO proteins was examined at the given times after the addition of rifampicin by northern blot analysis. As controls, FinP stability was examined in the absence of FinO, and hybridization was performed on RNA extracted from cells not harboring the F-plasmid.