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