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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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Related in: MedlinePlus

Conserved arginine patch on the rim of Hfq. (a) Electrostatic contact potentials were calculated on the surface of Hfq (1HK9) using PyMOL and viewed from the proximal side. Conserved arginines on the rim of Hfq (Supplementary Figure S1) are shown in detail. The disordered C-terminal 30 residues are not visible but extend from a point near the distal side of this basic patch. (b) lacZ reporter for regulation of rpoS translation by sRNAs was introduced into the E. coli chromosome under control of PBAD promoter. (c) β-galactosidase activity in strains containing sRNA expression plasmids or pLac vector, in the presence of endogenous (WT) Hfq, or Hfq containing rim mutations.
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gkt521-F1: Conserved arginine patch on the rim of Hfq. (a) Electrostatic contact potentials were calculated on the surface of Hfq (1HK9) using PyMOL and viewed from the proximal side. Conserved arginines on the rim of Hfq (Supplementary Figure S1) are shown in detail. The disordered C-terminal 30 residues are not visible but extend from a point near the distal side of this basic patch. (b) lacZ reporter for regulation of rpoS translation by sRNAs was introduced into the E. coli chromosome under control of PBAD promoter. (c) β-galactosidase activity in strains containing sRNA expression plasmids or pLac vector, in the presence of endogenous (WT) Hfq, or Hfq containing rim mutations.

Mentions: Arginines at positions 16, 17 and 19 of E. coli Hfq form a patch of positive charge on the outer rim of the Sm core, at the top of a shallow groove leading out from the RNA-binding site along the inner surface of the proximal face (Figure 1a) (14). Alignment of 384 Hfq sequences (Supplementary Figure S1) showed that R16 is nearly invariant within this sequence collection, whereas position 17 is usually R or K (Figure 1a). Position 19 is least conserved, but frequently R, K or N. The acidic E18 side chain, which is also moderately conserved, points away from the basic patch toward the distal face.Figure 1.


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

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

Conserved arginine patch on the rim of Hfq. (a) Electrostatic contact potentials were calculated on the surface of Hfq (1HK9) using PyMOL and viewed from the proximal side. Conserved arginines on the rim of Hfq (Supplementary Figure S1) are shown in detail. The disordered C-terminal 30 residues are not visible but extend from a point near the distal side of this basic patch. (b) lacZ reporter for regulation of rpoS translation by sRNAs was introduced into the E. coli chromosome under control of PBAD promoter. (c) β-galactosidase activity in strains containing sRNA expression plasmids or pLac vector, in the presence of endogenous (WT) Hfq, or Hfq containing rim mutations.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt521-F1: Conserved arginine patch on the rim of Hfq. (a) Electrostatic contact potentials were calculated on the surface of Hfq (1HK9) using PyMOL and viewed from the proximal side. Conserved arginines on the rim of Hfq (Supplementary Figure S1) are shown in detail. The disordered C-terminal 30 residues are not visible but extend from a point near the distal side of this basic patch. (b) lacZ reporter for regulation of rpoS translation by sRNAs was introduced into the E. coli chromosome under control of PBAD promoter. (c) β-galactosidase activity in strains containing sRNA expression plasmids or pLac vector, in the presence of endogenous (WT) Hfq, or Hfq containing rim mutations.
Mentions: Arginines at positions 16, 17 and 19 of E. coli Hfq form a patch of positive charge on the outer rim of the Sm core, at the top of a shallow groove leading out from the RNA-binding site along the inner surface of the proximal face (Figure 1a) (14). Alignment of 384 Hfq sequences (Supplementary Figure S1) showed that R16 is nearly invariant within this sequence collection, whereas position 17 is usually R or K (Figure 1a). Position 19 is least conserved, but frequently R, K or N. The acidic E18 side chain, which is also moderately conserved, points away from the basic patch toward the distal face.Figure 1.

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