Limits...
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.

Show MeSH

Related in: MedlinePlus

StpA mutant and domains. (A) Schematic representation of the different protein constructs used to specify the regions responsible for the RNA chaperone activity of StpA. The N-terminal (NH2–StpA) and the C-terminal domains (COOH–StpA) as well as a mutant with a glycine to valine change at position 126 (G126V–StpA) in the DNA-binding domain were prepared. (B) In splicing assays, 1.4 µM of the different protein constructs were tested for RNA chaperone activity with 0.5 pM of the sho-sho pre-mRNA. The graphs show the decrease of pre-mRNA during splicing.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1851640&req=5

Figure 6: StpA mutant and domains. (A) Schematic representation of the different protein constructs used to specify the regions responsible for the RNA chaperone activity of StpA. The N-terminal (NH2–StpA) and the C-terminal domains (COOH–StpA) as well as a mutant with a glycine to valine change at position 126 (G126V–StpA) in the DNA-binding domain were prepared. (B) In splicing assays, 1.4 µM of the different protein constructs were tested for RNA chaperone activity with 0.5 pM of the sho-sho pre-mRNA. The graphs show the decrease of pre-mRNA during splicing.

Mentions: Like its paralogue H-NS, StpA has a two-domain structure (40). The negatively charged N-terminal region is responsible for dimerization, whereas the positively charged C-terminus is needed for nucleic acid binding. The two domains are interconnected by a flexible linker region (Figure 6A) (42). The C-terminal domain had previously been reported to exert RNA annealing activity and also to promote trans-splicing in vitro (40). To map the RNA chaperone activity of StpA, we purified both domains separately (Figure 6A). We performed cis-splicing assays with the N-terminal and C-terminal domains separately and compared the activities to that of the full-length protein. We were able to confirm that the C-terminal domain shows RNA chaperone activity although to a lesser extent than the full-length protein. The N-terminal domain also showed RNA chaperone activity in the cis-splicing assay (Figure 6B and Table 2). The kobs value of the second phase is raised fourfold in the presence of NH2–StpA, suggesting that it can activate the population of molecules that is not induced by the full-length protein. Furthermore, from the X-ray crystal structure of the N-terminal domain of an H-NS homologue, it has recently been proposed that the N-terminal domain contributes to DNA recognition (42).Figure 6.


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 mutant and domains. (A) Schematic representation of the different protein constructs used to specify the regions responsible for the RNA chaperone activity of StpA. The N-terminal (NH2–StpA) and the C-terminal domains (COOH–StpA) as well as a mutant with a glycine to valine change at position 126 (G126V–StpA) in the DNA-binding domain were prepared. (B) In splicing assays, 1.4 µM of the different protein constructs were tested for RNA chaperone activity with 0.5 pM of the sho-sho pre-mRNA. The graphs show the decrease of pre-mRNA during splicing.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 6: StpA mutant and domains. (A) Schematic representation of the different protein constructs used to specify the regions responsible for the RNA chaperone activity of StpA. The N-terminal (NH2–StpA) and the C-terminal domains (COOH–StpA) as well as a mutant with a glycine to valine change at position 126 (G126V–StpA) in the DNA-binding domain were prepared. (B) In splicing assays, 1.4 µM of the different protein constructs were tested for RNA chaperone activity with 0.5 pM of the sho-sho pre-mRNA. The graphs show the decrease of pre-mRNA during splicing.
Mentions: Like its paralogue H-NS, StpA has a two-domain structure (40). The negatively charged N-terminal region is responsible for dimerization, whereas the positively charged C-terminus is needed for nucleic acid binding. The two domains are interconnected by a flexible linker region (Figure 6A) (42). The C-terminal domain had previously been reported to exert RNA annealing activity and also to promote trans-splicing in vitro (40). To map the RNA chaperone activity of StpA, we purified both domains separately (Figure 6A). We performed cis-splicing assays with the N-terminal and C-terminal domains separately and compared the activities to that of the full-length protein. We were able to confirm that the C-terminal domain shows RNA chaperone activity although to a lesser extent than the full-length protein. The N-terminal domain also showed RNA chaperone activity in the cis-splicing assay (Figure 6B and Table 2). The kobs value of the second phase is raised fourfold in the presence of NH2–StpA, suggesting that it can activate the population of molecules that is not induced by the full-length protein. Furthermore, from the X-ray crystal structure of the N-terminal domain of an H-NS homologue, it has recently been proposed that the N-terminal domain contributes to DNA recognition (42).Figure 6.

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