FIGURE 2.

FRET assay for S4-rRNA interactions. (A) DNA oligonucleotides complementary to 3′-extensions of 5′ domains (Supplemental Table S1) were tested for annealing by EMSA. Sample gels for Oligo 6 (left panel), Oligo 9 (center), and Oligo 5 (right). The 5′-Dom-h3E9 RNA adopts multiple conformations. 5′-Dom-h3E5 RNA and Oligo5 was selected for fluorescence experiments. (B) EMSA shows that protein S4 binds the extended RNA–DNA complex. (C) FRET upon addition of 10 nM S4-Cy5 to 5′-Dom-h3E5 RNA-h3-oligo-Cy3 complex (1.5 nM each in 20 mM MgCl₂) at 37°C. (D) Addition of unlabeled S4 (10 nM) at similar conditions shows no significant change in donor fluorescence. (E) Addition of acceptor-labeled protein S4 to h3Cy3 oligo shows no FRET signal.