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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 V1 cleavage of 5′- and 3′-end-labeled SLIIc in the absence and presence of various FinO constructs. (A) 15% urea-denaturing polyacrylamide gels showing the products of RNase V1 (0.01 U) cleavage reactions. The lanes OH and T1 represent the alkaline hydrolysis and RNase T1 cleavage of denatured SLIIc, respectively. SLIIc nucleotide positions are indicated at the right of the gels. Large and small arrowheads indicate major or minor cleavages by RNase V1 in the presence of FinO. Vertical brackets represent significant footprints on SLIIc RNA resulting from FinO protection of SLIIc from RNase V1 attack. The asterisk at position C10 marks very weak protection of the lower stem at the 5′-end of SLIIc. (B) Bar graphs showing the quantification of the footprint areas of the gels in (A). The left axis shows the degree of FinO protection from RNase V1 relative to the ‘No protein’ reaction. In black is FinO1–186 WT, white is FinO33–186 W36A, and in gray is FinO45–186. Data above the horizontal dashed rule in each graph represent significant protection (≥2-fold) by FinO. The black bars below the x-axis highlight the footprint.
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Figure 3: RNase V1 cleavage of 5′- and 3′-end-labeled SLIIc in the absence and presence of various FinO constructs. (A) 15% urea-denaturing polyacrylamide gels showing the products of RNase V1 (0.01 U) cleavage reactions. The lanes OH and T1 represent the alkaline hydrolysis and RNase T1 cleavage of denatured SLIIc, respectively. SLIIc nucleotide positions are indicated at the right of the gels. Large and small arrowheads indicate major or minor cleavages by RNase V1 in the presence of FinO. Vertical brackets represent significant footprints on SLIIc RNA resulting from FinO protection of SLIIc from RNase V1 attack. The asterisk at position C10 marks very weak protection of the lower stem at the 5′-end of SLIIc. (B) Bar graphs showing the quantification of the footprint areas of the gels in (A). The left axis shows the degree of FinO protection from RNase V1 relative to the ‘No protein’ reaction. In black is FinO1–186 WT, white is FinO33–186 W36A, and in gray is FinO45–186. Data above the horizontal dashed rule in each graph represent significant protection (≥2-fold) by FinO. The black bars below the x-axis highlight the footprint.

Mentions: To quantify the footprinting data, we first normalized the total counts in each FinO–RNA complex lane to the total counts of the ‘No Protein’ lane to account for lane loading discrepancies. Then for each band of interest, representing a position in the RNA, the fraction (f) of the total counts for that band was calculated (fp, i) where p is a FinO–RNA complex (either FinO, FinO33–186 W36A or FinO45–186), and i is the nucleotide position of the RNA. We also determined the fraction of the total counts for the ‘No Protein’ sample (fnp,i) at this position. Finally, we divide fnp,i by fp,i to get the protection factor which is defined as the magnitude by which the RNA was protected from RNase cleavage by each FinO. For each RNase cleavage experiment, two independent reactions were performed and loaded onto the same gel. The values in Figures 2B and 3B are an average of these two independent reactions. We decided on a protection value of two or greater to represent a significant footprint. This is shown in the figures as a horizontal rule across the graph.Figure 3.


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 V1 cleavage of 5′- and 3′-end-labeled SLIIc in the absence and presence of various FinO constructs. (A) 15% urea-denaturing polyacrylamide gels showing the products of RNase V1 (0.01 U) cleavage reactions. The lanes OH and T1 represent the alkaline hydrolysis and RNase T1 cleavage of denatured SLIIc, respectively. SLIIc nucleotide positions are indicated at the right of the gels. Large and small arrowheads indicate major or minor cleavages by RNase V1 in the presence of FinO. Vertical brackets represent significant footprints on SLIIc RNA resulting from FinO protection of SLIIc from RNase V1 attack. The asterisk at position C10 marks very weak protection of the lower stem at the 5′-end of SLIIc. (B) Bar graphs showing the quantification of the footprint areas of the gels in (A). The left axis shows the degree of FinO protection from RNase V1 relative to the ‘No protein’ reaction. In black is FinO1–186 WT, white is FinO33–186 W36A, and in gray is FinO45–186. Data above the horizontal dashed rule in each graph represent significant protection (≥2-fold) by FinO. The black bars below the x-axis highlight the footprint.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: RNase V1 cleavage of 5′- and 3′-end-labeled SLIIc in the absence and presence of various FinO constructs. (A) 15% urea-denaturing polyacrylamide gels showing the products of RNase V1 (0.01 U) cleavage reactions. The lanes OH and T1 represent the alkaline hydrolysis and RNase T1 cleavage of denatured SLIIc, respectively. SLIIc nucleotide positions are indicated at the right of the gels. Large and small arrowheads indicate major or minor cleavages by RNase V1 in the presence of FinO. Vertical brackets represent significant footprints on SLIIc RNA resulting from FinO protection of SLIIc from RNase V1 attack. The asterisk at position C10 marks very weak protection of the lower stem at the 5′-end of SLIIc. (B) Bar graphs showing the quantification of the footprint areas of the gels in (A). The left axis shows the degree of FinO protection from RNase V1 relative to the ‘No protein’ reaction. In black is FinO1–186 WT, white is FinO33–186 W36A, and in gray is FinO45–186. Data above the horizontal dashed rule in each graph represent significant protection (≥2-fold) by FinO. The black bars below the x-axis highlight the footprint.
Mentions: To quantify the footprinting data, we first normalized the total counts in each FinO–RNA complex lane to the total counts of the ‘No Protein’ lane to account for lane loading discrepancies. Then for each band of interest, representing a position in the RNA, the fraction (f) of the total counts for that band was calculated (fp, i) where p is a FinO–RNA complex (either FinO, FinO33–186 W36A or FinO45–186), and i is the nucleotide position of the RNA. We also determined the fraction of the total counts for the ‘No Protein’ sample (fnp,i) at this position. Finally, we divide fnp,i by fp,i to get the protection factor which is defined as the magnitude by which the RNA was protected from RNase cleavage by each FinO. For each RNase cleavage experiment, two independent reactions were performed and loaded onto the same gel. The values in Figures 2B and 3B are an average of these two independent reactions. We decided on a protection value of two or greater to represent a significant footprint. This is shown in the figures as a horizontal rule across the graph.Figure 3.

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