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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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Summary of RNase V1 and I cleavage reactions of SLII and SLIIc. (A) Secondary structures of SLII and SLIIc showing the results from the RNase cleavage reactions. Large and small black arrowheads denote strong and weak RNase V1 cleavages, respectively in the presence of the FinO constructs. Large and small open arrowheads denote strong and weak RNase I cleavages, respectively in the presence of FinO. Boxes indicate footprints where FinO protected the RNAs from RNase V1 cleavage. Dashed boxes indicate areas of protection from RNase I cleavage by FinO. An area where a ‘V’ resides indicates enhanced cleavage by RNase I in the presence of the FinO constructs. (B) Electrostatic potentials at the solvent accessible surface of FinO33–184, contoured at ±10 kT/e. Approximate surface locations of the six FinO side-chains known to cross-link to SLII are labeled and shown with semi-transparent circles.
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Figure 7: Summary of RNase V1 and I cleavage reactions of SLII and SLIIc. (A) Secondary structures of SLII and SLIIc showing the results from the RNase cleavage reactions. Large and small black arrowheads denote strong and weak RNase V1 cleavages, respectively in the presence of the FinO constructs. Large and small open arrowheads denote strong and weak RNase I cleavages, respectively in the presence of FinO. Boxes indicate footprints where FinO protected the RNAs from RNase V1 cleavage. Dashed boxes indicate areas of protection from RNase I cleavage by FinO. An area where a ‘V’ resides indicates enhanced cleavage by RNase I in the presence of the FinO constructs. (B) Electrostatic potentials at the solvent accessible surface of FinO33–184, contoured at ±10 kT/e. Approximate surface locations of the six FinO side-chains known to cross-link to SLII are labeled and shown with semi-transparent circles.

Mentions: A model of SLII including the 5′- and 3′-tails was initially constructed based on the NMR structure of the initiator tRNA anti-codon loop from yeast, a 21-nt RNA stem–loop (RCSB accession code 1SZY) (23). The nucleotides were mutated to correspond to the sequence of SLII using COOT (24) and the stem–loop geometry-regularized in CNS (25). A model for FinO45–186 was derived by removing residues 33–44 from the crystal structure (RCSB accession code 1DVO). The docking of SLII to FinO45–186 was performed using the chemical restraint-driven program HADDOCK2.0 (26,27). On the protein, active ambiguous air restraints (AIRs) were defined as residues found to form chemical cross-links with the RNA as determined in Ghetu et al. (14) (Lys46, Lys81, Arg121, Lys125, Arg165 and Lys176). Passive AIRs on SLII were those nucleotides determined by ribonuclease footprinting to be protected in the complex, namely nucleotides 7, 8 and 36–45 (numbering as SLII in Figure 7A). No passive AIRS were defined for FinO, and no active AIRs were defined for SLII. Default settings were used for most HADDOCK parameters. No AIRs were randomly removed during separate docking trials. The stem of SLII was further geometry-restrained to be an A-form helix with Watson–Crick base pairing. The 5′- and 3′-tails of SLII were defined as both semi- and fully flexible regions. Semi-flexible protein residues were automatically chosen by HADDOCK if they were within 5.0 Å of the RNA. A control experiment was carried out, in which 2500 docking trials used randomly defined AIRs with random removal of 50% of AIRs per trial. Filtering of all docked models against the SAXS data used the program CRYSOL (28) to calculate theoretical scattering curves and obtain values of RG and Dmax for the models. The theoretical scattering curves were fit to the experimental SAXS data using CRYSOL in the range 0 < s < 0.3 Å−1 to provide a goodness-of-fit value, χ2. The top 250 models, ordered on fit to the data, were subjected to pair-wise cluster analysis based on structural similarity. The RMSD cutoff of 13 Å was chosen as small as possible while still providing enough models in a cluster to be meaningful. The five clusters obtained had their members aligned using the maximum-likelihood superpositioning program THESEUS (29).


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)

Summary of RNase V1 and I cleavage reactions of SLII and SLIIc. (A) Secondary structures of SLII and SLIIc showing the results from the RNase cleavage reactions. Large and small black arrowheads denote strong and weak RNase V1 cleavages, respectively in the presence of the FinO constructs. Large and small open arrowheads denote strong and weak RNase I cleavages, respectively in the presence of FinO. Boxes indicate footprints where FinO protected the RNAs from RNase V1 cleavage. Dashed boxes indicate areas of protection from RNase I cleavage by FinO. An area where a ‘V’ resides indicates enhanced cleavage by RNase I in the presence of the FinO constructs. (B) Electrostatic potentials at the solvent accessible surface of FinO33–184, contoured at ±10 kT/e. Approximate surface locations of the six FinO side-chains known to cross-link to SLII are labeled and shown with semi-transparent circles.
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Related In: Results  -  Collection

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Figure 7: Summary of RNase V1 and I cleavage reactions of SLII and SLIIc. (A) Secondary structures of SLII and SLIIc showing the results from the RNase cleavage reactions. Large and small black arrowheads denote strong and weak RNase V1 cleavages, respectively in the presence of the FinO constructs. Large and small open arrowheads denote strong and weak RNase I cleavages, respectively in the presence of FinO. Boxes indicate footprints where FinO protected the RNAs from RNase V1 cleavage. Dashed boxes indicate areas of protection from RNase I cleavage by FinO. An area where a ‘V’ resides indicates enhanced cleavage by RNase I in the presence of the FinO constructs. (B) Electrostatic potentials at the solvent accessible surface of FinO33–184, contoured at ±10 kT/e. Approximate surface locations of the six FinO side-chains known to cross-link to SLII are labeled and shown with semi-transparent circles.
Mentions: A model of SLII including the 5′- and 3′-tails was initially constructed based on the NMR structure of the initiator tRNA anti-codon loop from yeast, a 21-nt RNA stem–loop (RCSB accession code 1SZY) (23). The nucleotides were mutated to correspond to the sequence of SLII using COOT (24) and the stem–loop geometry-regularized in CNS (25). A model for FinO45–186 was derived by removing residues 33–44 from the crystal structure (RCSB accession code 1DVO). The docking of SLII to FinO45–186 was performed using the chemical restraint-driven program HADDOCK2.0 (26,27). On the protein, active ambiguous air restraints (AIRs) were defined as residues found to form chemical cross-links with the RNA as determined in Ghetu et al. (14) (Lys46, Lys81, Arg121, Lys125, Arg165 and Lys176). Passive AIRs on SLII were those nucleotides determined by ribonuclease footprinting to be protected in the complex, namely nucleotides 7, 8 and 36–45 (numbering as SLII in Figure 7A). No passive AIRS were defined for FinO, and no active AIRs were defined for SLII. Default settings were used for most HADDOCK parameters. No AIRs were randomly removed during separate docking trials. The stem of SLII was further geometry-restrained to be an A-form helix with Watson–Crick base pairing. The 5′- and 3′-tails of SLII were defined as both semi- and fully flexible regions. Semi-flexible protein residues were automatically chosen by HADDOCK if they were within 5.0 Å of the RNA. A control experiment was carried out, in which 2500 docking trials used randomly defined AIRs with random removal of 50% of AIRs per trial. Filtering of all docked models against the SAXS data used the program CRYSOL (28) to calculate theoretical scattering curves and obtain values of RG and Dmax for the models. The theoretical scattering curves were fit to the experimental SAXS data using CRYSOL in the range 0 < s < 0.3 Å−1 to provide a goodness-of-fit value, χ2. The top 250 models, ordered on fit to the data, were subjected to pair-wise cluster analysis based on structural similarity. The RMSD cutoff of 13 Å was chosen as small as possible while still providing enough models in a cluster to be meaningful. The five clusters obtained had their members aligned using the maximum-likelihood superpositioning program THESEUS (29).

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