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. 2021 Aug 4;12:4696. doi: 10.1038/s41467-021-24964-2

Fig. 6. Puf6 ushers rRNA tertiary contact formation.

Fig. 6

a Tertiary contact between H22-H88 kissing loop (KL, in red) and H68 tip (GAAA in blue) within 25S rRNA (left; PDB ID: 4V7R), Mg2+ in green. Middle: tertiary contact between H22-H88 KL (in red) and H68 tip (GAAA in blue) within 28S rRNA (PDB ID: 4UG0) Right: knowledge-based structure prediction of the RNA construct (colored) matches well (RMSD = 1.6 Å) with the tertiary contact within 25S rRNA (gray). Dotted lines indicate poly-A linkers. b Events leading to tertiary contact formation (nucleolar particles, PDB ID: 6CB1 (ref. 6); NE1 and NE2 particles, PDB ID: 6YLX and 6YLY (ref. 8); nuclear particle, PDB ID: 3JCT (ref. 17)). Puf6 functions between NE2 and Nog2 particle, in dashed-line oval. c FRET efficiencies for 1 µM Cy3s (donor)/Cy5s (acceptor) labeled RNA: KL, KL-TLGAAA, and TLR-TLGAAA (cartoons above plots, Supplementary Table 3) were plotted against indicated K+ (left panel; 20 mM HEPES buffer) or Mg2+ (right panel; 20 mM HEPES, 100 mM K+) with (red circles) or without (black circles) 10 µM GB1-Puf6-PUM (161–656). d FRET increase upon addition of K+ (in blue), Mg2+ (in green), and 10 µM GB1-Puf6-PUM (in absence of Mg2+, in red) for KL, KL-TLGAAA and TLR-TLGAAA RNA constructs. Each independent experiment is presented (dots), and error bars indicate ±SD of n > 2 independent experiments. e UV thermal melting curves for KL-TLGAAA in 20 mM HEPES buffer containing 100 mM K+ (top) and 100 mM Mg2+ (bottom). Horizontal arrow: KL melts at a higher temperature in presence of Mg2+; vertical arrow: melting transition corresponding to KL-TLGAAA docking in presence of Mg2+. f Scheme for KL-TLGAAA formation in presence of K+, Mg2+, and Puf6. g In total, 2.5 nM Cy5-labeled H68- or H68-H88-H22-RNA was titrated with 0.16 nM to 20 µM Puf6-PUM(161–656). Fluorescence anisotropy was measured using a Tecan Safire II plate reader. Puf6 binding to H68 alone and to the KL-TLGAAA construct was KD ~ 10 µM and KD ~ 125 nM, respectively. Source data are provided as a Source Data file.