Xenbase Image ID: 127435

Figure 4. Determinants of Lin28:pre-let-7f interactions. (A) EMSAs with Xtr-pre-let-7f as a probe, mixed with increasing concentrations of protein (for XtrLin28b and XtrLin28b CSD+ZKD: 0, 0.4, 0.8, 1.6, 3.2, 6.4, 13, 26 µM; for XtrLin28b CSD and ZKD: 0, 0.8, 1.6, 3.2, 6.4, 13, 26, 51 µM). All EMSAs were performed using 1 nM α-32P-ATP-labeled RNA. (B) The terminal loop sequence and secondary structure of the Xtr-pre-let-7f mutants as predicted by Mfold (49). Regions that were mutated are shaded. (C) Results from EMSA experiments shown in (A). The binding affinity was scored according to the following dissociation constant ranges: +++++, 0.8–1.6 µM; ++++, 1.6–3.2 µM; +++, 3.2–6.4 µM; ++, 6.4–12.8 µM; +, 12.8–25.6 µM; −, >25.6 µM. (D and E) In vitro pre-miRNA processing reaction on 32P 5′-end-labeled Xtr-pre-let-7f. The indicated concentrations (in micromolar) of XtrLin28b, XtrLin28b RBDs, XtrLin28b CSD and XtrLin28b ZKD were added to 1 nM Xtr-pre-let-7f in the presence or absence of human Dicer. The samples were resolved on a 10% (w/v) denaturing PAGE and visualized by autoradiography. Image published in: Mayr F et al. (2012) © The Author(s) 2012. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license Larger Image Printer Friendly View

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