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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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RNase I overdigestion of 5′- and 3′-end-labeled SLII and SLIIc in absence and presence of various FinO constructs. In all experiments, the RNAs were digested with RNase I at a final concentration of 0.1 U/µl. (Ai) A 8% native EMSA showing binding reactions of 5′-32P-SLII with increasing amounts of wild-type FinO before the addition of RNase I. The final concentration of FinO WT in micromolar in each reaction is indicated on top of the gel. (Aii) Slice from a 15% urea-denaturing gel showing the products of the RNase I digest of 5′-32P-SLII at the 3′-end in the presence of increasing amounts of FinO. The lanes correspond to the binding reactions in (Ai). The lanes OH and T1 represent the alkaline hydrolysis and RNase T1 cleavage of denatured 5′-32P-SLII, respectively. SLII nucleotide positions are indicated at the right of the gel. (B) Slice from a 15% urea-denaturing gel showing the products of the RNase I digest of 5′-32P-SLIIc at the 3′-end in the absence and presence of various FinO constructs. Nucleotide position G39 is indicated next to the gel. (C) Slice from a 15% urea-denaturing gel showing the products of the RNase I digest of 3′-32P-SLIIc at the 5′-end in the absence and presence of various FinO constructs. SLIIc nucleotide positions are indicated next to the gel. (D) Slice from a 15% urea-denaturing gel showing the products of the RNase I digest of 3′-32P-SLII at the 5′-end in the absence and presence of various FinO constructs. Nucleotide position G5 is indicated at the right of the gel. For experiments in (B–D), a 1:1 molar ratio FinO-RNA complex was formed prior to exposure to RNase I.
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Figure 5: RNase I overdigestion of 5′- and 3′-end-labeled SLII and SLIIc in absence and presence of various FinO constructs. In all experiments, the RNAs were digested with RNase I at a final concentration of 0.1 U/µl. (Ai) A 8% native EMSA showing binding reactions of 5′-32P-SLII with increasing amounts of wild-type FinO before the addition of RNase I. The final concentration of FinO WT in micromolar in each reaction is indicated on top of the gel. (Aii) Slice from a 15% urea-denaturing gel showing the products of the RNase I digest of 5′-32P-SLII at the 3′-end in the presence of increasing amounts of FinO. The lanes correspond to the binding reactions in (Ai). The lanes OH and T1 represent the alkaline hydrolysis and RNase T1 cleavage of denatured 5′-32P-SLII, respectively. SLII nucleotide positions are indicated at the right of the gel. (B) Slice from a 15% urea-denaturing gel showing the products of the RNase I digest of 5′-32P-SLIIc at the 3′-end in the absence and presence of various FinO constructs. Nucleotide position G39 is indicated next to the gel. (C) Slice from a 15% urea-denaturing gel showing the products of the RNase I digest of 3′-32P-SLIIc at the 5′-end in the absence and presence of various FinO constructs. SLIIc nucleotide positions are indicated next to the gel. (D) Slice from a 15% urea-denaturing gel showing the products of the RNase I digest of 3′-32P-SLII at the 5′-end in the absence and presence of various FinO constructs. Nucleotide position G5 is indicated at the right of the gel. For experiments in (B–D), a 1:1 molar ratio FinO-RNA complex was formed prior to exposure to RNase I.

Mentions: To more clearly assess this protection, we carried out RNase I footprinting experiments at a much higher RNase I concentration, to enhance cleavage of the 3′-tail and reduce the amount of full-length RNA which would otherwise obscure the footprint. The clearest results were obtained for 5′-labeled SLII RNA (Figure 5A). Under these conditions, the 3′-tail was completely removed in the absence of FinO. Dramatic protection was observed with increasing FinO concentrations, suggesting a strong interaction between FinO and the 3′-tail. Less dramatic but significant protection was observed for the 3′-tail of SLIIc (Figure 5B). Similar results were also observed for FinO33–186 W36A and FinO45–186 (data not shown).Figure 5.


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)

RNase I overdigestion of 5′- and 3′-end-labeled SLII and SLIIc in absence and presence of various FinO constructs. In all experiments, the RNAs were digested with RNase I at a final concentration of 0.1 U/µl. (Ai) A 8% native EMSA showing binding reactions of 5′-32P-SLII with increasing amounts of wild-type FinO before the addition of RNase I. The final concentration of FinO WT in micromolar in each reaction is indicated on top of the gel. (Aii) Slice from a 15% urea-denaturing gel showing the products of the RNase I digest of 5′-32P-SLII at the 3′-end in the presence of increasing amounts of FinO. The lanes correspond to the binding reactions in (Ai). The lanes OH and T1 represent the alkaline hydrolysis and RNase T1 cleavage of denatured 5′-32P-SLII, respectively. SLII nucleotide positions are indicated at the right of the gel. (B) Slice from a 15% urea-denaturing gel showing the products of the RNase I digest of 5′-32P-SLIIc at the 3′-end in the absence and presence of various FinO constructs. Nucleotide position G39 is indicated next to the gel. (C) Slice from a 15% urea-denaturing gel showing the products of the RNase I digest of 3′-32P-SLIIc at the 5′-end in the absence and presence of various FinO constructs. SLIIc nucleotide positions are indicated next to the gel. (D) Slice from a 15% urea-denaturing gel showing the products of the RNase I digest of 3′-32P-SLII at the 5′-end in the absence and presence of various FinO constructs. Nucleotide position G5 is indicated at the right of the gel. For experiments in (B–D), a 1:1 molar ratio FinO-RNA complex was formed prior to exposure to RNase I.
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Figure 5: RNase I overdigestion of 5′- and 3′-end-labeled SLII and SLIIc in absence and presence of various FinO constructs. In all experiments, the RNAs were digested with RNase I at a final concentration of 0.1 U/µl. (Ai) A 8% native EMSA showing binding reactions of 5′-32P-SLII with increasing amounts of wild-type FinO before the addition of RNase I. The final concentration of FinO WT in micromolar in each reaction is indicated on top of the gel. (Aii) Slice from a 15% urea-denaturing gel showing the products of the RNase I digest of 5′-32P-SLII at the 3′-end in the presence of increasing amounts of FinO. The lanes correspond to the binding reactions in (Ai). The lanes OH and T1 represent the alkaline hydrolysis and RNase T1 cleavage of denatured 5′-32P-SLII, respectively. SLII nucleotide positions are indicated at the right of the gel. (B) Slice from a 15% urea-denaturing gel showing the products of the RNase I digest of 5′-32P-SLIIc at the 3′-end in the absence and presence of various FinO constructs. Nucleotide position G39 is indicated next to the gel. (C) Slice from a 15% urea-denaturing gel showing the products of the RNase I digest of 3′-32P-SLIIc at the 5′-end in the absence and presence of various FinO constructs. SLIIc nucleotide positions are indicated next to the gel. (D) Slice from a 15% urea-denaturing gel showing the products of the RNase I digest of 3′-32P-SLII at the 5′-end in the absence and presence of various FinO constructs. Nucleotide position G5 is indicated at the right of the gel. For experiments in (B–D), a 1:1 molar ratio FinO-RNA complex was formed prior to exposure to RNase I.
Mentions: To more clearly assess this protection, we carried out RNase I footprinting experiments at a much higher RNase I concentration, to enhance cleavage of the 3′-tail and reduce the amount of full-length RNA which would otherwise obscure the footprint. The clearest results were obtained for 5′-labeled SLII RNA (Figure 5A). Under these conditions, the 3′-tail was completely removed in the absence of FinO. Dramatic protection was observed with increasing FinO concentrations, suggesting a strong interaction between FinO and the 3′-tail. Less dramatic but significant protection was observed for the 3′-tail of SLIIc (Figure 5B). Similar results were also observed for FinO33–186 W36A and FinO45–186 (data not shown).Figure 5.

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
Related in: MedlinePlus