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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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Overview of constructs used in the study. (A and B) The secondary structures of traJ mRNA and FinP, respectively. The ribosomal binding site and start codon of traJ mRNA are boxed. The derivative RNAs of focus, SLIIc and SLII, are shown in bold and numbered accordingly. (C) The secondary structure SLII LV1 RNA, used to form a complex with the FinO45–186 in the SAXS experiments. SLII LV1 deviates from wild-type SLII in its loop region, which is shown in bold. (D) The crystal structure of FinO26–186 highlighting the protein constructs used in the experiments. The W36A mutation is shown as a ball on the structure. The N-terminal 32 amino acids were not observed due to disorder in the structure and are drawn in (dashed section). Below is a scaled linear representation of the primary structure of FinO showing where each construct begins. The shades of gray correspond to the crystal structure coloring. (E) A representative 8% native PAGE demonstrating each of the FinO constructs in a 1:1 molar complex with 5′-32P-SLII.
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Figure 1: Overview of constructs used in the study. (A and B) The secondary structures of traJ mRNA and FinP, respectively. The ribosomal binding site and start codon of traJ mRNA are boxed. The derivative RNAs of focus, SLIIc and SLII, are shown in bold and numbered accordingly. (C) The secondary structure SLII LV1 RNA, used to form a complex with the FinO45–186 in the SAXS experiments. SLII LV1 deviates from wild-type SLII in its loop region, which is shown in bold. (D) The crystal structure of FinO26–186 highlighting the protein constructs used in the experiments. The W36A mutation is shown as a ball on the structure. The N-terminal 32 amino acids were not observed due to disorder in the structure and are drawn in (dashed section). Below is a scaled linear representation of the primary structure of FinO showing where each construct begins. The shades of gray correspond to the crystal structure coloring. (E) A representative 8% native PAGE demonstrating each of the FinO constructs in a 1:1 molar complex with 5′-32P-SLII.

Mentions: Repression of conjugation relies on a plasmid-encoded antisense RNA system called FinOP. The antisense RNA, FinP, is complementary to the 5′-UTR of traJ, the transcriptional activator of the tra operon (3–6) (Figure 1A and B). Binding of FinP to the traJ mRNA occludes the ribosomal binding site, inhibiting traJ translation and preventing tra operon expression via H-NS de-silencing (7). FinP alone, however, is unable to repress conjugation, in part because it is rapidly degraded by RNaseE, a component of the E. coli RNA degradosome (8,9). FinP requires a second plasmid factor, the FinO protein, which binds FinP and stabilizes it against degradation (8,10). FinO also binds the traJ 5′-UTR, and facilitates FinP–traJ RNA interactions, to ultimately repress conjugation 100- to 1000-fold.Figure 1.


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)

Overview of constructs used in the study. (A and B) The secondary structures of traJ mRNA and FinP, respectively. The ribosomal binding site and start codon of traJ mRNA are boxed. The derivative RNAs of focus, SLIIc and SLII, are shown in bold and numbered accordingly. (C) The secondary structure SLII LV1 RNA, used to form a complex with the FinO45–186 in the SAXS experiments. SLII LV1 deviates from wild-type SLII in its loop region, which is shown in bold. (D) The crystal structure of FinO26–186 highlighting the protein constructs used in the experiments. The W36A mutation is shown as a ball on the structure. The N-terminal 32 amino acids were not observed due to disorder in the structure and are drawn in (dashed section). Below is a scaled linear representation of the primary structure of FinO showing where each construct begins. The shades of gray correspond to the crystal structure coloring. (E) A representative 8% native PAGE demonstrating each of the FinO constructs in a 1:1 molar complex with 5′-32P-SLII.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Overview of constructs used in the study. (A and B) The secondary structures of traJ mRNA and FinP, respectively. The ribosomal binding site and start codon of traJ mRNA are boxed. The derivative RNAs of focus, SLIIc and SLII, are shown in bold and numbered accordingly. (C) The secondary structure SLII LV1 RNA, used to form a complex with the FinO45–186 in the SAXS experiments. SLII LV1 deviates from wild-type SLII in its loop region, which is shown in bold. (D) The crystal structure of FinO26–186 highlighting the protein constructs used in the experiments. The W36A mutation is shown as a ball on the structure. The N-terminal 32 amino acids were not observed due to disorder in the structure and are drawn in (dashed section). Below is a scaled linear representation of the primary structure of FinO showing where each construct begins. The shades of gray correspond to the crystal structure coloring. (E) A representative 8% native PAGE demonstrating each of the FinO constructs in a 1:1 molar complex with 5′-32P-SLII.
Mentions: Repression of conjugation relies on a plasmid-encoded antisense RNA system called FinOP. The antisense RNA, FinP, is complementary to the 5′-UTR of traJ, the transcriptional activator of the tra operon (3–6) (Figure 1A and B). Binding of FinP to the traJ mRNA occludes the ribosomal binding site, inhibiting traJ translation and preventing tra operon expression via H-NS de-silencing (7). FinP alone, however, is unable to repress conjugation, in part because it is rapidly degraded by RNaseE, a component of the E. coli RNA degradosome (8,9). FinP requires a second plasmid factor, the FinO protein, which binds FinP and stabilizes it against degradation (8,10). FinO also binds the traJ 5′-UTR, and facilitates FinP–traJ RNA interactions, to ultimately repress conjugation 100- to 1000-fold.Figure 1.

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