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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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In vitro selection of StpA- and Hfq-binding RNAs from a genomic E. coli library. The percentage of recovered RNA in relation to the input RNA is shown for both proteins for eight consecutive selection cycles. For the Hfq, used here as a positive control, RNAs are enriched and increasing amounts of the input RNA were recovered. No StpA-binding RNAs could be selected and recovery remained at background levels throughout the procedure.
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Figure 4: In vitro selection of StpA- and Hfq-binding RNAs from a genomic E. coli library. The percentage of recovered RNA in relation to the input RNA is shown for both proteins for eight consecutive selection cycles. For the Hfq, used here as a positive control, RNAs are enriched and increasing amounts of the input RNA were recovered. No StpA-binding RNAs could be selected and recovery remained at background levels throughout the procedure.

Mentions: We further asked the question if there are any endogenous StpA-binding RNAs in E. coli. In order to identify potential candidates, we performed a genomic selection. A genomic library of E. coli that contains overlapping fragments of approximately 50–500 nt in length was constructed. This library was used for several rounds of selection to enrich RNAs that bind to StpA. We used two different binding buffers for parallel selections. Buffer A was chosen to resemble physiological conditions and buffer B was adopted from Brescia et al. (39) as the authors studied the binding of rpoS mRNA and the non-coding RNA DsrA to the StpA homologue H-NS under these conditions. For the binding reaction, we used RNA (10 µM) in a tenfold molar excess over protein (1 µM). The protein Hfq, which is known to bind several RNAs of E. coli, served as a positive control for the selection procedure, and RNAs binding to Hfq were selected using buffer A. Results of the selections are shown in Figure 4. As expected, Hfq-binding RNAs were enriched over five consecutive selection cycles and up to 2.7% of the input RNA could be recovered. It should be noted that due to the tenfold molar excess of RNA over protein the maximal recovery rate is 10% assuming a single binding site. However, no StpA-binding RNAs could be enriched, neither for buffer A (data not shown) nor for buffer B and the recovery of bound RNAs remained at background levels throughout the selection procedure. We therefore assume that StpA has no specific RNA target in E. coli, at least under the tested conditions. This result stresses the non-specificity of StpA–RNA interactions and suggests that StpA is a general RNA chaperone.Figure 4.


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)

In vitro selection of StpA- and Hfq-binding RNAs from a genomic E. coli library. The percentage of recovered RNA in relation to the input RNA is shown for both proteins for eight consecutive selection cycles. For the Hfq, used here as a positive control, RNAs are enriched and increasing amounts of the input RNA were recovered. No StpA-binding RNAs could be selected and recovery remained at background levels throughout the procedure.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1851640&req=5

Figure 4: In vitro selection of StpA- and Hfq-binding RNAs from a genomic E. coli library. The percentage of recovered RNA in relation to the input RNA is shown for both proteins for eight consecutive selection cycles. For the Hfq, used here as a positive control, RNAs are enriched and increasing amounts of the input RNA were recovered. No StpA-binding RNAs could be selected and recovery remained at background levels throughout the procedure.
Mentions: We further asked the question if there are any endogenous StpA-binding RNAs in E. coli. In order to identify potential candidates, we performed a genomic selection. A genomic library of E. coli that contains overlapping fragments of approximately 50–500 nt in length was constructed. This library was used for several rounds of selection to enrich RNAs that bind to StpA. We used two different binding buffers for parallel selections. Buffer A was chosen to resemble physiological conditions and buffer B was adopted from Brescia et al. (39) as the authors studied the binding of rpoS mRNA and the non-coding RNA DsrA to the StpA homologue H-NS under these conditions. For the binding reaction, we used RNA (10 µM) in a tenfold molar excess over protein (1 µM). The protein Hfq, which is known to bind several RNAs of E. coli, served as a positive control for the selection procedure, and RNAs binding to Hfq were selected using buffer A. Results of the selections are shown in Figure 4. As expected, Hfq-binding RNAs were enriched over five consecutive selection cycles and up to 2.7% of the input RNA could be recovered. It should be noted that due to the tenfold molar excess of RNA over protein the maximal recovery rate is 10% assuming a single binding site. However, no StpA-binding RNAs could be enriched, neither for buffer A (data not shown) nor for buffer B and the recovery of bound RNAs remained at background levels throughout the selection procedure. We therefore assume that StpA has no specific RNA target in E. coli, at least under the tested conditions. This result stresses the non-specificity of StpA–RNA interactions and suggests that StpA is a general RNA chaperone.Figure 4.

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