Limits...
Structural basis of nucleic acid binding by Nicotiana tabacum glycine-rich RNA-binding protein: implications for its RNA chaperone function.

Khan F, Daniëls MA, Folkers GE, Boelens R, Saqlan Naqvi SM, van Ingen H - Nucleic Acids Res. (2014)

Bottom Line: A HADDOCK model of the NtRRM-RNA complex, based on NMR chemical shift and NOE data, shows that nucleic acid binding results from a combination of stacking and electrostatic interactions with conserved RRM residues.Finally, DNA melting experiments demonstrate that NtGR-RBP1 is more efficient in melting CTG containing nucleic acids than isolated NtRRM.Together, our study supports the model that self-association of GR-RBPs by the glycine-rich region results in cooperative unfolding of non-native substrate structures, thereby enhancing its chaperone function.

View Article: PubMed Central - PubMed

Affiliation: NMR Spectroscopy Research Group, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands Department of Biochemistry, PMAS Agriculture University Rawalpindi, 46300 Rawalpindi, Pakistan.

Show MeSH

Related in: MedlinePlus

Glycine-rich region stimulates higher-order complex formation and dsDNA unfolding (A) Representative DNA binding experiment by EMSA for the binding of 0, 0.7, 2, 7, 20 μM NtRRM or NtGR-RBP1 to a single or double ssDNA binding element (ssP1 or ss-dP1). Dashed lines indicate the positions of the wells and the free mobilities of ssP1 and ss-dP1, and serve to guide the eye. The arrows indicate lanes of interest with clear band shifts. Several lanes show a smeared, asymmetric band appearance caused by significant dissociation during electrophoresis. The selection box for quantification of the free DNA probe is indicated in white on the first lane. (B) 1D traces of the lanes with 20 μM NtRRM or NtGR-RBP1 added to ssP1 show a small but reproducible and concentration-dependent shifts in mobility. (C) Quantification of the fraction of bound ssDNA for the ss-dP1 probe at the indicated concentration of NtGR-RBP1 and NtRRM, the line represents the calculated binding curve based on three independent experiments. (D) UV melting curves of indicated oligonucleotides in presence of 3 M equivalents of NtGR-RBP1, NtRRM or BSA. Absorbance (at 260 nm) versus temperature curves in 20 mM KPi, 100 mM KCl, 1 mM BME at pH 7.0. Temperatures (°C) at the transition midpoint, Tm, are indicated for the free DNA probes, with changes in Tm listed in the presence of the three proteins.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4117745&req=5

Figure 8: Glycine-rich region stimulates higher-order complex formation and dsDNA unfolding (A) Representative DNA binding experiment by EMSA for the binding of 0, 0.7, 2, 7, 20 μM NtRRM or NtGR-RBP1 to a single or double ssDNA binding element (ssP1 or ss-dP1). Dashed lines indicate the positions of the wells and the free mobilities of ssP1 and ss-dP1, and serve to guide the eye. The arrows indicate lanes of interest with clear band shifts. Several lanes show a smeared, asymmetric band appearance caused by significant dissociation during electrophoresis. The selection box for quantification of the free DNA probe is indicated in white on the first lane. (B) 1D traces of the lanes with 20 μM NtRRM or NtGR-RBP1 added to ssP1 show a small but reproducible and concentration-dependent shifts in mobility. (C) Quantification of the fraction of bound ssDNA for the ss-dP1 probe at the indicated concentration of NtGR-RBP1 and NtRRM, the line represents the calculated binding curve based on three independent experiments. (D) UV melting curves of indicated oligonucleotides in presence of 3 M equivalents of NtGR-RBP1, NtRRM or BSA. Absorbance (at 260 nm) versus temperature curves in 20 mM KPi, 100 mM KCl, 1 mM BME at pH 7.0. Temperatures (°C) at the transition midpoint, Tm, are indicated for the free DNA probes, with changes in Tm listed in the presence of the three proteins.

Mentions: To further evaluate the role of the GR in nucleic acid binding, we performed electrophoretic mobility shift assays (EMSA) for both NtRRM and NtGR-RBP1 using a ssDNA probe (ssP1) having the CTG-containing mRNA-binding site of homologue AtGR-RBP7 (34) in either a single (13-nt) or double (26-nt) copy (designated as ssP1 or ss-dP1, Figure 8A). Due to the small size of the single probe ssP1, only very limited shift in mobility for both proteins was obtained even at high gel concentrations (see also Figure 8B), precluding estimation of the apparent binding affinity. Nucleic acid binding is nevertheless apparent from the loss of free probe for the RRM domain and from the slight shift and band smearing for the full-length protein. For the longer ss-dP1 probe, a clear mobility shift is obtained for NtRRM at the two highest protein concentrations, together with significant band smearing at the highest RRM concentration. Both observations indicate nucleic acid binding, as expected. Using the loss of free probe to estimate the fraction bound, an apparent dissociation constant KD,app in the low micromolar range was estimated (∼10 μM, Figure 8C). Similarly, a KD,app of ∼5 μM was estimated for NtGR-RBP1, in correspondence with the NMR-based finding that RRM domain and full-length protein have comparable affinities for ssDNA. Strikingly, for NtGR-RBP1, a very slow migrating band is visible at the highest protein concentration, after an initial smaller shift. This suggests formation of a higher-order complex. While this complex travelled only slightly through the gel, substantial migration of this band could be established at lower gel concentrations, but at the expense of resolution for the initial smaller shift (data not shown). The diffuse appearance of the band indicates substantial dissociation during electrophoresis. Importantly, no discrete stable higher order complex is visible at the highest concentration of NtRRM. There, band smearing above the shifted probe suggests formation of transient higher-order complexes that dissociate during electrophoresis. The lower apparent binding affinity and lack of stable higher-order complex formation for NtRRM suggest that the GR contributes to DNA binding affinity, possibly by stimulating dimerisation or multimerisation of NtGR-RBP1.


Structural basis of nucleic acid binding by Nicotiana tabacum glycine-rich RNA-binding protein: implications for its RNA chaperone function.

Khan F, Daniëls MA, Folkers GE, Boelens R, Saqlan Naqvi SM, van Ingen H - Nucleic Acids Res. (2014)

Glycine-rich region stimulates higher-order complex formation and dsDNA unfolding (A) Representative DNA binding experiment by EMSA for the binding of 0, 0.7, 2, 7, 20 μM NtRRM or NtGR-RBP1 to a single or double ssDNA binding element (ssP1 or ss-dP1). Dashed lines indicate the positions of the wells and the free mobilities of ssP1 and ss-dP1, and serve to guide the eye. The arrows indicate lanes of interest with clear band shifts. Several lanes show a smeared, asymmetric band appearance caused by significant dissociation during electrophoresis. The selection box for quantification of the free DNA probe is indicated in white on the first lane. (B) 1D traces of the lanes with 20 μM NtRRM or NtGR-RBP1 added to ssP1 show a small but reproducible and concentration-dependent shifts in mobility. (C) Quantification of the fraction of bound ssDNA for the ss-dP1 probe at the indicated concentration of NtGR-RBP1 and NtRRM, the line represents the calculated binding curve based on three independent experiments. (D) UV melting curves of indicated oligonucleotides in presence of 3 M equivalents of NtGR-RBP1, NtRRM or BSA. Absorbance (at 260 nm) versus temperature curves in 20 mM KPi, 100 mM KCl, 1 mM BME at pH 7.0. Temperatures (°C) at the transition midpoint, Tm, are indicated for the free DNA probes, with changes in Tm listed in the presence of the three proteins.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4117745&req=5

Figure 8: Glycine-rich region stimulates higher-order complex formation and dsDNA unfolding (A) Representative DNA binding experiment by EMSA for the binding of 0, 0.7, 2, 7, 20 μM NtRRM or NtGR-RBP1 to a single or double ssDNA binding element (ssP1 or ss-dP1). Dashed lines indicate the positions of the wells and the free mobilities of ssP1 and ss-dP1, and serve to guide the eye. The arrows indicate lanes of interest with clear band shifts. Several lanes show a smeared, asymmetric band appearance caused by significant dissociation during electrophoresis. The selection box for quantification of the free DNA probe is indicated in white on the first lane. (B) 1D traces of the lanes with 20 μM NtRRM or NtGR-RBP1 added to ssP1 show a small but reproducible and concentration-dependent shifts in mobility. (C) Quantification of the fraction of bound ssDNA for the ss-dP1 probe at the indicated concentration of NtGR-RBP1 and NtRRM, the line represents the calculated binding curve based on three independent experiments. (D) UV melting curves of indicated oligonucleotides in presence of 3 M equivalents of NtGR-RBP1, NtRRM or BSA. Absorbance (at 260 nm) versus temperature curves in 20 mM KPi, 100 mM KCl, 1 mM BME at pH 7.0. Temperatures (°C) at the transition midpoint, Tm, are indicated for the free DNA probes, with changes in Tm listed in the presence of the three proteins.
Mentions: To further evaluate the role of the GR in nucleic acid binding, we performed electrophoretic mobility shift assays (EMSA) for both NtRRM and NtGR-RBP1 using a ssDNA probe (ssP1) having the CTG-containing mRNA-binding site of homologue AtGR-RBP7 (34) in either a single (13-nt) or double (26-nt) copy (designated as ssP1 or ss-dP1, Figure 8A). Due to the small size of the single probe ssP1, only very limited shift in mobility for both proteins was obtained even at high gel concentrations (see also Figure 8B), precluding estimation of the apparent binding affinity. Nucleic acid binding is nevertheless apparent from the loss of free probe for the RRM domain and from the slight shift and band smearing for the full-length protein. For the longer ss-dP1 probe, a clear mobility shift is obtained for NtRRM at the two highest protein concentrations, together with significant band smearing at the highest RRM concentration. Both observations indicate nucleic acid binding, as expected. Using the loss of free probe to estimate the fraction bound, an apparent dissociation constant KD,app in the low micromolar range was estimated (∼10 μM, Figure 8C). Similarly, a KD,app of ∼5 μM was estimated for NtGR-RBP1, in correspondence with the NMR-based finding that RRM domain and full-length protein have comparable affinities for ssDNA. Strikingly, for NtGR-RBP1, a very slow migrating band is visible at the highest protein concentration, after an initial smaller shift. This suggests formation of a higher-order complex. While this complex travelled only slightly through the gel, substantial migration of this band could be established at lower gel concentrations, but at the expense of resolution for the initial smaller shift (data not shown). The diffuse appearance of the band indicates substantial dissociation during electrophoresis. Importantly, no discrete stable higher order complex is visible at the highest concentration of NtRRM. There, band smearing above the shifted probe suggests formation of transient higher-order complexes that dissociate during electrophoresis. The lower apparent binding affinity and lack of stable higher-order complex formation for NtRRM suggest that the GR contributes to DNA binding affinity, possibly by stimulating dimerisation or multimerisation of NtGR-RBP1.

Bottom Line: A HADDOCK model of the NtRRM-RNA complex, based on NMR chemical shift and NOE data, shows that nucleic acid binding results from a combination of stacking and electrostatic interactions with conserved RRM residues.Finally, DNA melting experiments demonstrate that NtGR-RBP1 is more efficient in melting CTG containing nucleic acids than isolated NtRRM.Together, our study supports the model that self-association of GR-RBPs by the glycine-rich region results in cooperative unfolding of non-native substrate structures, thereby enhancing its chaperone function.

View Article: PubMed Central - PubMed

Affiliation: NMR Spectroscopy Research Group, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands Department of Biochemistry, PMAS Agriculture University Rawalpindi, 46300 Rawalpindi, Pakistan.

Show MeSH
Related in: MedlinePlus