Limits...
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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Limited RNase I digestion of 5′- and 3′-end-labeled SLII and SLIIc in the absence and presence of various FinO constructs. Products of the RNase I (0.01 U/µl final concentration) cleavage reactions were resolved on 15% urea-denaturing polyacrylamide gels. The radiolabeled RNA construct is noted below each of the gels. The lanes OH and T1 represent the alkaline hydrolysis and RNase T1 cleavage of denatured SLII or SLIIc, respectively. The RNA nucleotide positions are indicated at the left of the gels. Large and small arrowheads indicate major or minor cleavages by RNase I in the presence of FinO while the vertical bracket indicates protection from RNase I.
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Figure 4: Limited RNase I digestion of 5′- and 3′-end-labeled SLII and SLIIc in the absence and presence of various FinO constructs. Products of the RNase I (0.01 U/µl final concentration) cleavage reactions were resolved on 15% urea-denaturing polyacrylamide gels. The radiolabeled RNA construct is noted below each of the gels. The lanes OH and T1 represent the alkaline hydrolysis and RNase T1 cleavage of denatured SLII or SLIIc, respectively. The RNA nucleotide positions are indicated at the left of the gels. Large and small arrowheads indicate major or minor cleavages by RNase I in the presence of FinO while the vertical bracket indicates protection from RNase I.

Mentions: It was previously shown that deletion of the seven nucleotide loop from SLII does not affect the affinity of FinO binding, suggesting the loop plays little if any role in specific, high affinity interactions with FinO (15). We used RNase I (34) footprinting to directly probe for interactions between FinO and single-stranded areas of SLII or SLIIc. Figure 4 shows the results for the limited RNase I digestion of SLII and SLIIc both free, and in a 1:1 complex with the three FinO constructs. For both RNAs, intense cleavage was observed, as expected, at the single-stranded loop regions, and FinO binding consistently resulted in a significant enhancement in this cleavage.Figure 4.


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)

Limited RNase I digestion of 5′- and 3′-end-labeled SLII and SLIIc in the absence and presence of various FinO constructs. Products of the RNase I (0.01 U/µl final concentration) cleavage reactions were resolved on 15% urea-denaturing polyacrylamide gels. The radiolabeled RNA construct is noted below each of the gels. The lanes OH and T1 represent the alkaline hydrolysis and RNase T1 cleavage of denatured SLII or SLIIc, respectively. The RNA nucleotide positions are indicated at the left of the gels. Large and small arrowheads indicate major or minor cleavages by RNase I in the presence of FinO while the vertical bracket indicates protection from RNase I.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Limited RNase I digestion of 5′- and 3′-end-labeled SLII and SLIIc in the absence and presence of various FinO constructs. Products of the RNase I (0.01 U/µl final concentration) cleavage reactions were resolved on 15% urea-denaturing polyacrylamide gels. The radiolabeled RNA construct is noted below each of the gels. The lanes OH and T1 represent the alkaline hydrolysis and RNase T1 cleavage of denatured SLII or SLIIc, respectively. The RNA nucleotide positions are indicated at the left of the gels. Large and small arrowheads indicate major or minor cleavages by RNase I in the presence of FinO while the vertical bracket indicates protection from RNase I.
Mentions: It was previously shown that deletion of the seven nucleotide loop from SLII does not affect the affinity of FinO binding, suggesting the loop plays little if any role in specific, high affinity interactions with FinO (15). We used RNase I (34) footprinting to directly probe for interactions between FinO and single-stranded areas of SLII or SLIIc. Figure 4 shows the results for the limited RNase I digestion of SLII and SLIIc both free, and in a 1:1 complex with the three FinO constructs. For both RNAs, intense cleavage was observed, as expected, at the single-stranded loop regions, and FinO binding consistently resulted in a significant enhancement in this cleavage.Figure 4.

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