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RNA chaperone activity and RNA-binding properties of the E. coli protein StpA.

Mayer O, Rajkowitsch L, Lorenz C, Konrat R, Schroeder R - Nucleic Acids Res. (2007)

Bottom Line: A mutant variant of the protein, with a glycine to valine change in the nucleic-acid-binding domain, displays weaker RNA binding but higher RNA chaperone activity.This suggests that the RNA chaperone activity of StpA results from weak and transient interactions rather than from tight binding to RNA.We further discuss the role that structural disorder in proteins may play in chaperoning RNA folding, using bioinformatic sequence analysis tools, and provide evidence for the importance of conformational disorder and local structural preformation of chaperone nucleic-acid-binding sites.

View Article: PubMed Central - PubMed

Affiliation: Max F. Perutz Laboratories, University of Vienna, Dr Bohrgasse 9/5, A-1030 Vienna, Austria.

ABSTRACT
The E. coli protein StpA has RNA annealing and strand displacement activities and it promotes folding of RNAs by loosening their structures. To understand the mode of action of StpA, we analysed the relationship of its RNA chaperone activity to its RNA-binding properties. For acceleration of annealing of two short RNAs, StpA binds both molecules simultaneously, showing that annealing is promoted by crowding. StpA binds weakly to RNA with a preference for unstructured molecules. Binding of StpA to RNA is strongly dependent on the ionic strength, suggesting that the interactions are mainly electrostatic. A mutant variant of the protein, with a glycine to valine change in the nucleic-acid-binding domain, displays weaker RNA binding but higher RNA chaperone activity. This suggests that the RNA chaperone activity of StpA results from weak and transient interactions rather than from tight binding to RNA. We further discuss the role that structural disorder in proteins may play in chaperoning RNA folding, using bioinformatic sequence analysis tools, and provide evidence for the importance of conformational disorder and local structural preformation of chaperone nucleic-acid-binding sites.

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Related in: MedlinePlus

RNA-binding assays (A) Different RNA constructs were incubated with increasing amounts of protein (1 pM to 4 µM). The td ribozyme construct lacks exon sequences and misses 7 nt of the 5′ end of the intron and 5 nt at the 3′ end; (B) Influence of mono- and divalent metal ions on the binding behaviour of the RNA chaperone to td ribozyme RNA. Increasing amounts of mono- and divalent ions lead to a drastic drop in the binding efficiency of StpA.
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Figure 3: RNA-binding assays (A) Different RNA constructs were incubated with increasing amounts of protein (1 pM to 4 µM). The td ribozyme construct lacks exon sequences and misses 7 nt of the 5′ end of the intron and 5 nt at the 3′ end; (B) Influence of mono- and divalent metal ions on the binding behaviour of the RNA chaperone to td ribozyme RNA. Increasing amounts of mono- and divalent ions lead to a drastic drop in the binding efficiency of StpA.

Mentions: To test how well StpA binds to differently structured RNAs, we prepared three types of the td RNA: (1) the mature mRNA without the intron sequences as an example of a mostly unstructured RNA, (2) the sho-sho-type pre-mRNA with short exon sequences as an example of a mixed RNA with a structured core but with unstructured flanking sequences and (3) the ribozyme core as an example of a compactly folded RNA. Folding of this construct is well characterized and it folds into a homogenous population of molecules (37,38). As can be seen in Figure 3A, none of these RNAs bind efficiently to 4 µM StpA. Under these conditions, 38% of the mRNA, 16% of the pre-mRNA and only trace amounts of the ribozyme are bound to StpA. This clearly demonstrates that StpA preferentially binds to unstructured RNAs. The dissociation constants are 0.58 μM for the intronless mRNA and 0.73 μM for the sho-sho pre-mRNA construct. The K1/2 for the ribozyme construct could not be unambiguously determined due to inefficient binding. At 1.4 µM, the previously observed optimal protein concentration for RNA chaperone activity, binding is significant although only a small fraction of the RNA molecules is bound to the protein.Figure 3.


RNA chaperone activity and RNA-binding properties of the E. coli protein StpA.

Mayer O, Rajkowitsch L, Lorenz C, Konrat R, Schroeder R - Nucleic Acids Res. (2007)

RNA-binding assays (A) Different RNA constructs were incubated with increasing amounts of protein (1 pM to 4 µM). The td ribozyme construct lacks exon sequences and misses 7 nt of the 5′ end of the intron and 5 nt at the 3′ end; (B) Influence of mono- and divalent metal ions on the binding behaviour of the RNA chaperone to td ribozyme RNA. Increasing amounts of mono- and divalent ions lead to a drastic drop in the binding efficiency of StpA.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851640&req=5

Figure 3: RNA-binding assays (A) Different RNA constructs were incubated with increasing amounts of protein (1 pM to 4 µM). The td ribozyme construct lacks exon sequences and misses 7 nt of the 5′ end of the intron and 5 nt at the 3′ end; (B) Influence of mono- and divalent metal ions on the binding behaviour of the RNA chaperone to td ribozyme RNA. Increasing amounts of mono- and divalent ions lead to a drastic drop in the binding efficiency of StpA.
Mentions: To test how well StpA binds to differently structured RNAs, we prepared three types of the td RNA: (1) the mature mRNA without the intron sequences as an example of a mostly unstructured RNA, (2) the sho-sho-type pre-mRNA with short exon sequences as an example of a mixed RNA with a structured core but with unstructured flanking sequences and (3) the ribozyme core as an example of a compactly folded RNA. Folding of this construct is well characterized and it folds into a homogenous population of molecules (37,38). As can be seen in Figure 3A, none of these RNAs bind efficiently to 4 µM StpA. Under these conditions, 38% of the mRNA, 16% of the pre-mRNA and only trace amounts of the ribozyme are bound to StpA. This clearly demonstrates that StpA preferentially binds to unstructured RNAs. The dissociation constants are 0.58 μM for the intronless mRNA and 0.73 μM for the sho-sho pre-mRNA construct. The K1/2 for the ribozyme construct could not be unambiguously determined due to inefficient binding. At 1.4 µM, the previously observed optimal protein concentration for RNA chaperone activity, binding is significant although only a small fraction of the RNA molecules is bound to the protein.Figure 3.

Bottom Line: A mutant variant of the protein, with a glycine to valine change in the nucleic-acid-binding domain, displays weaker RNA binding but higher RNA chaperone activity.This suggests that the RNA chaperone activity of StpA results from weak and transient interactions rather than from tight binding to RNA.We further discuss the role that structural disorder in proteins may play in chaperoning RNA folding, using bioinformatic sequence analysis tools, and provide evidence for the importance of conformational disorder and local structural preformation of chaperone nucleic-acid-binding sites.

View Article: PubMed Central - PubMed

Affiliation: Max F. Perutz Laboratories, University of Vienna, Dr Bohrgasse 9/5, A-1030 Vienna, Austria.

ABSTRACT
The E. coli protein StpA has RNA annealing and strand displacement activities and it promotes folding of RNAs by loosening their structures. To understand the mode of action of StpA, we analysed the relationship of its RNA chaperone activity to its RNA-binding properties. For acceleration of annealing of two short RNAs, StpA binds both molecules simultaneously, showing that annealing is promoted by crowding. StpA binds weakly to RNA with a preference for unstructured molecules. Binding of StpA to RNA is strongly dependent on the ionic strength, suggesting that the interactions are mainly electrostatic. A mutant variant of the protein, with a glycine to valine change in the nucleic-acid-binding domain, displays weaker RNA binding but higher RNA chaperone activity. This suggests that the RNA chaperone activity of StpA results from weak and transient interactions rather than from tight binding to RNA. We further discuss the role that structural disorder in proteins may play in chaperoning RNA folding, using bioinformatic sequence analysis tools, and provide evidence for the importance of conformational disorder and local structural preformation of chaperone nucleic-acid-binding sites.

Show MeSH
Related in: MedlinePlus