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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

StpA-induced splicing of the td pre-mRNA. (A) Splicing assay of the sho-sho RNA in the presence of increasing amounts of StpA. The reactions were performed at 37°C as described in experimental procedures. Increasing the amount of the RNA chaperone leads to an increase of the fast-reacting RNA population with a peak activity at 1.4 µM StpA. Higher StpA concentrations again reduce the activity. (B) Comparison of the splicing behaviour of the sho-sho RNA in the absence and after addition of StpA to the splicing reaction after 10 min of incubation. StpA causes a burst of activity immediately after addition.
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Figure 2: StpA-induced splicing of the td pre-mRNA. (A) Splicing assay of the sho-sho RNA in the presence of increasing amounts of StpA. The reactions were performed at 37°C as described in experimental procedures. Increasing the amount of the RNA chaperone leads to an increase of the fast-reacting RNA population with a peak activity at 1.4 µM StpA. Higher StpA concentrations again reduce the activity. (B) Comparison of the splicing behaviour of the sho-sho RNA in the absence and after addition of StpA to the splicing reaction after 10 min of incubation. StpA causes a burst of activity immediately after addition.

Mentions: We studied the effect of different concentrations of StpA on folding and splicing of the sho-sho RNA construct. Splicing experiments were performed in the presence of increasing amounts of StpA. The presence of StpA leads to an increase of RNA molecules in the fast splicing population (Figure 2). The higher the concentration of StpA the more RNA molecules are in a splicing competent conformation and undergo the splicing reaction fast. A maximum of about 64% of the RNAs can be shifted to the correctly folded conformation in the presence of 1.4 µM StpA. This protein concentration seems optimal for chaperone activity. Increasing the protein concentration higher than 1.4 µM leads to a less efficient promotion of folding. StpA does not interfere with the splicing reaction rates. The values for the kobs of the fast-reacting fraction of molecules stay around 0.4 min−1. Values for the second, slower rate remain about two orders of magnitude lower, at 5 × 10−3 min−1 (Table 1).Figure 2.


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)

StpA-induced splicing of the td pre-mRNA. (A) Splicing assay of the sho-sho RNA in the presence of increasing amounts of StpA. The reactions were performed at 37°C as described in experimental procedures. Increasing the amount of the RNA chaperone leads to an increase of the fast-reacting RNA population with a peak activity at 1.4 µM StpA. Higher StpA concentrations again reduce the activity. (B) Comparison of the splicing behaviour of the sho-sho RNA in the absence and after addition of StpA to the splicing reaction after 10 min of incubation. StpA causes a burst of activity immediately after addition.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 2: StpA-induced splicing of the td pre-mRNA. (A) Splicing assay of the sho-sho RNA in the presence of increasing amounts of StpA. The reactions were performed at 37°C as described in experimental procedures. Increasing the amount of the RNA chaperone leads to an increase of the fast-reacting RNA population with a peak activity at 1.4 µM StpA. Higher StpA concentrations again reduce the activity. (B) Comparison of the splicing behaviour of the sho-sho RNA in the absence and after addition of StpA to the splicing reaction after 10 min of incubation. StpA causes a burst of activity immediately after addition.
Mentions: We studied the effect of different concentrations of StpA on folding and splicing of the sho-sho RNA construct. Splicing experiments were performed in the presence of increasing amounts of StpA. The presence of StpA leads to an increase of RNA molecules in the fast splicing population (Figure 2). The higher the concentration of StpA the more RNA molecules are in a splicing competent conformation and undergo the splicing reaction fast. A maximum of about 64% of the RNAs can be shifted to the correctly folded conformation in the presence of 1.4 µM StpA. This protein concentration seems optimal for chaperone activity. Increasing the protein concentration higher than 1.4 µM leads to a less efficient promotion of folding. StpA does not interfere with the splicing reaction rates. The values for the kobs of the fast-reacting fraction of molecules stay around 0.4 min−1. Values for the second, slower rate remain about two orders of magnitude lower, at 5 × 10−3 min−1 (Table 1).Figure 2.

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