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Mapping interactions between the RNA chaperone FinO and its RNA targets.

Arthur DC, Edwards RA, Tsutakawa S, Tainer JA, Frost LS, Glover JN - Nucleic Acids Res. (2011)

Bottom Line: The collective results allow the generation of an energy-minimized model of the FinO-SLII complex, consistent with small-angle X-ray scattering data.The repression complex model was constrained using previously reported cross-linking data and newly developed footprinting results.Together, these data lead us to propose a model of how FinO mediates FinP/traJ mRNA pairing to down regulate bacterial conjugation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Alberta, Edmonton, AB T6G 2H7, Canada.

ABSTRACT
Bacterial conjugation is regulated by two-component repression comprising the antisense RNA FinP, and its protein co-factor FinO. FinO mediates base-pairing of FinP to the 5'-untranslated region (UTR) of traJ mRNA, which leads to translational inhibition of the transcriptional activator TraJ and subsequent down regulation of conjugation genes. Yet, little is known about how FinO binds to its RNA targets or how this interaction facilitates FinP and traJ mRNA pairing. Here, we use solution methods to determine how FinO binds specifically to its minimal high affinity target, FinP stem-loop II (SLII), and its complement SLIIc from traJ mRNA. Ribonuclease footprinting reveals that FinO contacts the base of the stem and the 3' single-stranded tails of these RNAs. The phosphorylation or oxidation of the 3'-nucleotide blocks FinO binding, suggesting FinO binds the 3'-hydroxyl of its RNA targets. The collective results allow the generation of an energy-minimized model of the FinO-SLII complex, consistent with small-angle X-ray scattering data. The repression complex model was constrained using previously reported cross-linking data and newly developed footprinting results. Together, these data lead us to propose a model of how FinO mediates FinP/traJ mRNA pairing to down regulate bacterial conjugation.

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Combining HADDOCK and SAXS to model FinO45–186: SLII. Pair-wise RMSD cluster analysis was performed on the top 250 models ordered on χ2. Five clusters were obtained using an RMSD cutoff of 13 Å. (A) Model complexes from the best cluster (mean χ2 for nine members, 4.1) are shown in gray. The average structure for this cluster as calculated using THESEUS is represented with red spheres. (B) The average structures for all five clusters are shown superimposed. The average structure for the best cluster shown in (A) is shown as a red ribbon, and that of a significant structural outlier in white.
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Figure 8: Combining HADDOCK and SAXS to model FinO45–186: SLII. Pair-wise RMSD cluster analysis was performed on the top 250 models ordered on χ2. Five clusters were obtained using an RMSD cutoff of 13 Å. (A) Model complexes from the best cluster (mean χ2 for nine members, 4.1) are shown in gray. The average structure for this cluster as calculated using THESEUS is represented with red spheres. (B) The average structures for all five clusters are shown superimposed. The average structure for the best cluster shown in (A) is shown as a red ribbon, and that of a significant structural outlier in white.

Mentions: For comparison, HADDOCK was run using randomly selected AIRs between residues and nucleotides containing >20% relative accessibility to generate 2500 models. This random-AIR model set had a mean χ2 of 17.5, significantly worse than that of the restrained model set, with the single best-fit to the experimental scattering of 2.1. Although this best-fit randomly restrained model slightly improves the fit to the SAXS data relative to the best-fit restrained model, inspection shows a poor fit to the biochemical data. The best 250 RNA–protein complex models, ordered on χ2 were subjected to RMSD-based pair-wise cluster analysis. Five clusters (Figure 8) met the criteria of having a pair-wise RMSD cutoff less than 13 Å and containing at least six members. In every case, the RNA docked to the side of FinO containing the positively charged patch and residues Arg121, Lys125 and Arg165. Alignment of the stem–loop was also consistent amongst all clusters with the base of the RNA closer to the globular portion of FinO and the stem-loop generally coincident with the N-terminal helical extension. A representative model from one of these clusters was chosen to illustrate the satisfaction of a number of the programmed distance restraints and the general orientation of the RNA stem (Figure 9A) while still providing a reasonable fit to the SAXS data (Figure 9B). Nucleotides C7 and G8 are partially buried against the N-terminal α-helix of FinO and the 3′-tail extends along the positively charged face of FinO within contact distance of residues Arg121, Lys125 and Arg165. The fit of this particular model to the experimental data, χ2 of 4.2, is comparable to the mean χ2 of 4.6 for all 39 models in the five clusters.Figure 8.


Mapping interactions between the RNA chaperone FinO and its RNA targets.

Arthur DC, Edwards RA, Tsutakawa S, Tainer JA, Frost LS, Glover JN - Nucleic Acids Res. (2011)

Combining HADDOCK and SAXS to model FinO45–186: SLII. Pair-wise RMSD cluster analysis was performed on the top 250 models ordered on χ2. Five clusters were obtained using an RMSD cutoff of 13 Å. (A) Model complexes from the best cluster (mean χ2 for nine members, 4.1) are shown in gray. The average structure for this cluster as calculated using THESEUS is represented with red spheres. (B) The average structures for all five clusters are shown superimposed. The average structure for the best cluster shown in (A) is shown as a red ribbon, and that of a significant structural outlier in white.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105414&req=5

Figure 8: Combining HADDOCK and SAXS to model FinO45–186: SLII. Pair-wise RMSD cluster analysis was performed on the top 250 models ordered on χ2. Five clusters were obtained using an RMSD cutoff of 13 Å. (A) Model complexes from the best cluster (mean χ2 for nine members, 4.1) are shown in gray. The average structure for this cluster as calculated using THESEUS is represented with red spheres. (B) The average structures for all five clusters are shown superimposed. The average structure for the best cluster shown in (A) is shown as a red ribbon, and that of a significant structural outlier in white.
Mentions: For comparison, HADDOCK was run using randomly selected AIRs between residues and nucleotides containing >20% relative accessibility to generate 2500 models. This random-AIR model set had a mean χ2 of 17.5, significantly worse than that of the restrained model set, with the single best-fit to the experimental scattering of 2.1. Although this best-fit randomly restrained model slightly improves the fit to the SAXS data relative to the best-fit restrained model, inspection shows a poor fit to the biochemical data. The best 250 RNA–protein complex models, ordered on χ2 were subjected to RMSD-based pair-wise cluster analysis. Five clusters (Figure 8) met the criteria of having a pair-wise RMSD cutoff less than 13 Å and containing at least six members. In every case, the RNA docked to the side of FinO containing the positively charged patch and residues Arg121, Lys125 and Arg165. Alignment of the stem–loop was also consistent amongst all clusters with the base of the RNA closer to the globular portion of FinO and the stem-loop generally coincident with the N-terminal helical extension. A representative model from one of these clusters was chosen to illustrate the satisfaction of a number of the programmed distance restraints and the general orientation of the RNA stem (Figure 9A) while still providing a reasonable fit to the SAXS data (Figure 9B). Nucleotides C7 and G8 are partially buried against the N-terminal α-helix of FinO and the 3′-tail extends along the positively charged face of FinO within contact distance of residues Arg121, Lys125 and Arg165. The fit of this particular model to the experimental data, χ2 of 4.2, is comparable to the mean χ2 of 4.6 for all 39 models in the five clusters.Figure 8.

Bottom Line: The collective results allow the generation of an energy-minimized model of the FinO-SLII complex, consistent with small-angle X-ray scattering data.The repression complex model was constrained using previously reported cross-linking data and newly developed footprinting results.Together, these data lead us to propose a model of how FinO mediates FinP/traJ mRNA pairing to down regulate bacterial conjugation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Alberta, Edmonton, AB T6G 2H7, Canada.

ABSTRACT
Bacterial conjugation is regulated by two-component repression comprising the antisense RNA FinP, and its protein co-factor FinO. FinO mediates base-pairing of FinP to the 5'-untranslated region (UTR) of traJ mRNA, which leads to translational inhibition of the transcriptional activator TraJ and subsequent down regulation of conjugation genes. Yet, little is known about how FinO binds to its RNA targets or how this interaction facilitates FinP and traJ mRNA pairing. Here, we use solution methods to determine how FinO binds specifically to its minimal high affinity target, FinP stem-loop II (SLII), and its complement SLIIc from traJ mRNA. Ribonuclease footprinting reveals that FinO contacts the base of the stem and the 3' single-stranded tails of these RNAs. The phosphorylation or oxidation of the 3'-nucleotide blocks FinO binding, suggesting FinO binds the 3'-hydroxyl of its RNA targets. The collective results allow the generation of an energy-minimized model of the FinO-SLII complex, consistent with small-angle X-ray scattering data. The repression complex model was constrained using previously reported cross-linking data and newly developed footprinting results. Together, these data lead us to propose a model of how FinO mediates FinP/traJ mRNA pairing to down regulate bacterial conjugation.

Show MeSH