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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 SLII in the absence and presence of various FinO constructs. (A) 15% urea-denaturing polyacrylamide gels showing the products of RNase V1 (0.001 U/µl final concentration) cleavage reactions. M10 and M15 are synthesized SLII RNA markers of 10 and 15 nucleotides in length (see ‘Materials and Methods’ section). The lanes OH and T1 represent the alkaline hydrolysis and RNase T1 cleavage of denatured SLII, respectively. SLII 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 SLII RNA resulting from FinO protection of SLII from RNase V1 attack. (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. The shift in the footprint when 5′- and 3′-end-labeling are compared is likely due to the effect of adding pCp to the 3′-end of the RNA when 3′-end-labeling. In Figure 6, we demonstrate that this subtly alters protein binding.
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Figure 2: RNase V1 cleavage of 5′- and 3′-end-labeled SLII in the absence and presence of various FinO constructs. (A) 15% urea-denaturing polyacrylamide gels showing the products of RNase V1 (0.001 U/µl final concentration) cleavage reactions. M10 and M15 are synthesized SLII RNA markers of 10 and 15 nucleotides in length (see ‘Materials and Methods’ section). The lanes OH and T1 represent the alkaline hydrolysis and RNase T1 cleavage of denatured SLII, respectively. SLII 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 SLII RNA resulting from FinO protection of SLII from RNase V1 attack. (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. The shift in the footprint when 5′- and 3′-end-labeling are compared is likely due to the effect of adding pCp to the 3′-end of the RNA when 3′-end-labeling. In Figure 6, we demonstrate that this subtly alters protein binding.

Mentions: We used two different methods to assign the RNase V1 cleavage products which, due to a 3′-OH, run slower on a denaturing gel. First, for the 5′-32P SLII RNase V1 cleavage assays, we chemically synthesized short RNA markers which had the same sequence as SLII and had a 3′-OH. SLII markers were 10 (5′-GACAGUCGAU-3′) and 15 (5′-GACAGUCGAUGCAGG-3′) nucleotides in length (Figure 2, left). The oligomers were synthesized, purified and labeled in the same manner as SLII and SLIIc (see above). The other method used T4 polynucleotide kinase, in the absence of ATP, to remove the 3′-phosphate from the RNase T1 and alkaline hydrolysis products (19).Figure 2.


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 SLII in the absence and presence of various FinO constructs. (A) 15% urea-denaturing polyacrylamide gels showing the products of RNase V1 (0.001 U/µl final concentration) cleavage reactions. M10 and M15 are synthesized SLII RNA markers of 10 and 15 nucleotides in length (see ‘Materials and Methods’ section). The lanes OH and T1 represent the alkaline hydrolysis and RNase T1 cleavage of denatured SLII, respectively. SLII 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 SLII RNA resulting from FinO protection of SLII from RNase V1 attack. (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. The shift in the footprint when 5′- and 3′-end-labeling are compared is likely due to the effect of adding pCp to the 3′-end of the RNA when 3′-end-labeling. In Figure 6, we demonstrate that this subtly alters protein binding.
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Related In: Results  -  Collection

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Figure 2: RNase V1 cleavage of 5′- and 3′-end-labeled SLII in the absence and presence of various FinO constructs. (A) 15% urea-denaturing polyacrylamide gels showing the products of RNase V1 (0.001 U/µl final concentration) cleavage reactions. M10 and M15 are synthesized SLII RNA markers of 10 and 15 nucleotides in length (see ‘Materials and Methods’ section). The lanes OH and T1 represent the alkaline hydrolysis and RNase T1 cleavage of denatured SLII, respectively. SLII 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 SLII RNA resulting from FinO protection of SLII from RNase V1 attack. (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. The shift in the footprint when 5′- and 3′-end-labeling are compared is likely due to the effect of adding pCp to the 3′-end of the RNA when 3′-end-labeling. In Figure 6, we demonstrate that this subtly alters protein binding.
Mentions: We used two different methods to assign the RNase V1 cleavage products which, due to a 3′-OH, run slower on a denaturing gel. First, for the 5′-32P SLII RNase V1 cleavage assays, we chemically synthesized short RNA markers which had the same sequence as SLII and had a 3′-OH. SLII markers were 10 (5′-GACAGUCGAU-3′) and 15 (5′-GACAGUCGAUGCAGG-3′) nucleotides in length (Figure 2, left). The oligomers were synthesized, purified and labeled in the same manner as SLII and SLIIc (see above). The other method used T4 polynucleotide kinase, in the absence of ATP, to remove the 3′-phosphate from the RNase T1 and alkaline hydrolysis products (19).Figure 2.

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