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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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Basic rim patch is required for annealing U-rich and A-rich substrates. (a) RNA oligonucleotides for Hfq annealing assays. Base pairing of a molecular beacon (blue) with oligo CA increases FAM fluorescence. Oligo CA contains a non-specific target region (gray) and 3′ A18 Hfq-binding site (green). (b) Annealing rates were measured by stopped-flow fluorescence in TNK buffer at 30°C, using 50 nM beacon (rMBDss), 50 nM target RNA and 0–5000 nM Hfq monomer. Observed rate constants (average of five trials) are plotted against Hfq concentration. (c) Oligo CU contains the non-specific target region and a 3′ U6 Hfq-binding site (orange). (d) Observed annealing rates for oligo CU as in (b).
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gkt521-F4: Basic rim patch is required for annealing U-rich and A-rich substrates. (a) RNA oligonucleotides for Hfq annealing assays. Base pairing of a molecular beacon (blue) with oligo CA increases FAM fluorescence. Oligo CA contains a non-specific target region (gray) and 3′ A18 Hfq-binding site (green). (b) Annealing rates were measured by stopped-flow fluorescence in TNK buffer at 30°C, using 50 nM beacon (rMBDss), 50 nM target RNA and 0–5000 nM Hfq monomer. Observed rate constants (average of five trials) are plotted against Hfq concentration. (c) Oligo CU contains the non-specific target region and a 3′ U6 Hfq-binding site (orange). (d) Observed annealing rates for oligo CU as in (b).

Mentions: We next used engineered oligonucleotides to dissect how the arginine patch functions in sRNA–mRNA annealing. The annealing rates were measured by stopped-flow spectroscopy, using an RNA molecular beacon that becomes more fluorescent when based paired with the target RNA (Figure 4a). When the target RNA had a 3′ A18 tail that binds the distal face (CA), WT Hfq accelerated annealing up to 30 times (black; Figure 4b). However, Hfq:R16A increased the rate of base pairing no more than six times above the no Hfq background (red; Figure 4b). A similar difference was observed when the target had a U6 tail (CU; Figure 4c and d), although the annealing rate was lower for both proteins because the U6 tail prevents release of the target from the proximal face of Hfq (30). The sensitivity of these U6 and A18 substrates to proximal and distal face mutations confirmed they bind opposite surfaces of Hfq as expected (30).Figure 4.


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

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

Basic rim patch is required for annealing U-rich and A-rich substrates. (a) RNA oligonucleotides for Hfq annealing assays. Base pairing of a molecular beacon (blue) with oligo CA increases FAM fluorescence. Oligo CA contains a non-specific target region (gray) and 3′ A18 Hfq-binding site (green). (b) Annealing rates were measured by stopped-flow fluorescence in TNK buffer at 30°C, using 50 nM beacon (rMBDss), 50 nM target RNA and 0–5000 nM Hfq monomer. Observed rate constants (average of five trials) are plotted against Hfq concentration. (c) Oligo CU contains the non-specific target region and a 3′ U6 Hfq-binding site (orange). (d) Observed annealing rates for oligo CU as in (b).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3753642&req=5

gkt521-F4: Basic rim patch is required for annealing U-rich and A-rich substrates. (a) RNA oligonucleotides for Hfq annealing assays. Base pairing of a molecular beacon (blue) with oligo CA increases FAM fluorescence. Oligo CA contains a non-specific target region (gray) and 3′ A18 Hfq-binding site (green). (b) Annealing rates were measured by stopped-flow fluorescence in TNK buffer at 30°C, using 50 nM beacon (rMBDss), 50 nM target RNA and 0–5000 nM Hfq monomer. Observed rate constants (average of five trials) are plotted against Hfq concentration. (c) Oligo CU contains the non-specific target region and a 3′ U6 Hfq-binding site (orange). (d) Observed annealing rates for oligo CU as in (b).
Mentions: We next used engineered oligonucleotides to dissect how the arginine patch functions in sRNA–mRNA annealing. The annealing rates were measured by stopped-flow spectroscopy, using an RNA molecular beacon that becomes more fluorescent when based paired with the target RNA (Figure 4a). When the target RNA had a 3′ A18 tail that binds the distal face (CA), WT Hfq accelerated annealing up to 30 times (black; Figure 4b). However, Hfq:R16A increased the rate of base pairing no more than six times above the no Hfq background (red; Figure 4b). A similar difference was observed when the target had a U6 tail (CU; Figure 4c and d), although the annealing rate was lower for both proteins because the U6 tail prevents release of the target from the proximal face of Hfq (30). The sensitivity of these U6 and A18 substrates to proximal and distal face mutations confirmed they bind opposite surfaces of Hfq as expected (30).Figure 4.

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