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Conserved arginines on the rim of Hfq catalyze base pair formation and exchange.

Panja S, Schu DJ, Woodson SA - Nucleic Acids Res. (2013)

Bottom Line: Here, we show that conserved arginines on the outer rim of the hexamer that are known to interact with sRNA bodies are required for Hfq's chaperone activity.Stopped-flow FRET and fluorescence anisotropy show that complementary RNAs transiently form a ternary complex with Hfq, but the RNAs are not released as a double helix in the absence of rim arginines.We propose that the arginine patch overcomes entropic and electrostatic barriers to helix nucleation and constitutes the active site for Hfq's chaperone function.

View Article: PubMed Central - PubMed

Affiliation: T.C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA and Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD 20892-5430, USA.

ABSTRACT
The Sm-like protein Hfq is required for gene regulation by small RNAs (sRNAs) in bacteria and facilitates base pairing between sRNAs and their mRNA targets. The proximal and distal faces of the Hfq hexamer specifically bind sRNA and mRNA targets, but they do not explain how Hfq accelerates the formation and exchange of RNA base pairs. Here, we show that conserved arginines on the outer rim of the hexamer that are known to interact with sRNA bodies are required for Hfq's chaperone activity. Mutations in the arginine patch lower the ability of Hfq to act in sRNA regulation of rpoS translation and eliminate annealing of natural sRNAs or unstructured oligonucleotides, without preventing binding to either the proximal or distal face. Stopped-flow FRET and fluorescence anisotropy show that complementary RNAs transiently form a ternary complex with Hfq, but the RNAs are not released as a double helix in the absence of rim arginines. RNAs bound to either face of Hfq quench the fluorescence of a tryptophan adjacent to the arginine patch, demonstrating that the rim can simultaneously engage two RNA strands. We propose that the arginine patch overcomes entropic and electrostatic barriers to helix nucleation and constitutes the active site for Hfq's chaperone function.

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Rim mutants cannot facilitate annealing between sRNA and mRNA. (a) Annealing of 32P-labeled rpoS323, 200 nM sRNA and 1 mM Hfq was measured by native gel mobility shifts (Supplementary Figure S5). (b) Observed rate constants for formation of the rpoS•sRNA•Hfq ternary complex for Hfq rim mutants. Gray, no Hfq; black striping, WT Hfq; red, R16A; blue, R16K; dark cyan, R19A; magenta, R16A:R17A:R19D (TM).
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gkt521-F2: Rim mutants cannot facilitate annealing between sRNA and mRNA. (a) Annealing of 32P-labeled rpoS323, 200 nM sRNA and 1 mM Hfq was measured by native gel mobility shifts (Supplementary Figure S5). (b) Observed rate constants for formation of the rpoS•sRNA•Hfq ternary complex for Hfq rim mutants. Gray, no Hfq; black striping, WT Hfq; red, R16A; blue, R16K; dark cyan, R19A; magenta, R16A:R17A:R19D (TM).

Mentions: Native gel mobility shift experiments (Supplementary Figure S4) showed that the R16A rim mutation had little effect on binding of Hfq to a fragment of the rpoS mRNA leader (rpoS323; Table 1), which interacts with the distal face of Hfq through an (AAN)4 and A6 motif (19,40) (Figure 2a). Binding of DsrA and RprA sRNA was weakened about three times (Table 1), whereas the largest change was a four-fold increase in the dissociation constant for ArcZ56 (Table 1), consistent with reduced co-immunoprecipitation of DsrA and ArcZ by Hfq:R16A (34). We used the processed 56 nt form of ArcZ sRNA for these experiments because full-length ArcZ does not anneal with rpoS mRNA in vitro (8). The triple mutant bound DsrA about as tightly as the R16A single mutant (Table 1). From these and further results described later in the text, we concluded that the rim arginines contribute to sRNA interactions but are not required for stable binding of sRNAs or mRNAs.Figure 2.


Conserved arginines on the rim of Hfq catalyze base pair formation and exchange.

Panja S, Schu DJ, Woodson SA - Nucleic Acids Res. (2013)

Rim mutants cannot facilitate annealing between sRNA and mRNA. (a) Annealing of 32P-labeled rpoS323, 200 nM sRNA and 1 mM Hfq was measured by native gel mobility shifts (Supplementary Figure S5). (b) Observed rate constants for formation of the rpoS•sRNA•Hfq ternary complex for Hfq rim mutants. Gray, no Hfq; black striping, WT Hfq; red, R16A; blue, R16K; dark cyan, R19A; magenta, R16A:R17A:R19D (TM).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753642&req=5

gkt521-F2: Rim mutants cannot facilitate annealing between sRNA and mRNA. (a) Annealing of 32P-labeled rpoS323, 200 nM sRNA and 1 mM Hfq was measured by native gel mobility shifts (Supplementary Figure S5). (b) Observed rate constants for formation of the rpoS•sRNA•Hfq ternary complex for Hfq rim mutants. Gray, no Hfq; black striping, WT Hfq; red, R16A; blue, R16K; dark cyan, R19A; magenta, R16A:R17A:R19D (TM).
Mentions: Native gel mobility shift experiments (Supplementary Figure S4) showed that the R16A rim mutation had little effect on binding of Hfq to a fragment of the rpoS mRNA leader (rpoS323; Table 1), which interacts with the distal face of Hfq through an (AAN)4 and A6 motif (19,40) (Figure 2a). Binding of DsrA and RprA sRNA was weakened about three times (Table 1), whereas the largest change was a four-fold increase in the dissociation constant for ArcZ56 (Table 1), consistent with reduced co-immunoprecipitation of DsrA and ArcZ by Hfq:R16A (34). We used the processed 56 nt form of ArcZ sRNA for these experiments because full-length ArcZ does not anneal with rpoS mRNA in vitro (8). The triple mutant bound DsrA about as tightly as the R16A single mutant (Table 1). From these and further results described later in the text, we concluded that the rim arginines contribute to sRNA interactions but are not required for stable binding of sRNAs or mRNAs.Figure 2.

Bottom Line: Here, we show that conserved arginines on the outer rim of the hexamer that are known to interact with sRNA bodies are required for Hfq's chaperone activity.Stopped-flow FRET and fluorescence anisotropy show that complementary RNAs transiently form a ternary complex with Hfq, but the RNAs are not released as a double helix in the absence of rim arginines.We propose that the arginine patch overcomes entropic and electrostatic barriers to helix nucleation and constitutes the active site for Hfq's chaperone function.

View Article: PubMed Central - PubMed

Affiliation: T.C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA and Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD 20892-5430, USA.

ABSTRACT
The Sm-like protein Hfq is required for gene regulation by small RNAs (sRNAs) in bacteria and facilitates base pairing between sRNAs and their mRNA targets. The proximal and distal faces of the Hfq hexamer specifically bind sRNA and mRNA targets, but they do not explain how Hfq accelerates the formation and exchange of RNA base pairs. Here, we show that conserved arginines on the outer rim of the hexamer that are known to interact with sRNA bodies are required for Hfq's chaperone activity. Mutations in the arginine patch lower the ability of Hfq to act in sRNA regulation of rpoS translation and eliminate annealing of natural sRNAs or unstructured oligonucleotides, without preventing binding to either the proximal or distal face. Stopped-flow FRET and fluorescence anisotropy show that complementary RNAs transiently form a ternary complex with Hfq, but the RNAs are not released as a double helix in the absence of rim arginines. RNAs bound to either face of Hfq quench the fluorescence of a tryptophan adjacent to the arginine patch, demonstrating that the rim can simultaneously engage two RNA strands. We propose that the arginine patch overcomes entropic and electrostatic barriers to helix nucleation and constitutes the active site for Hfq's chaperone function.

Show MeSH
Related in: MedlinePlus