FIGURE 2.

Protein binding increases base accessibility in the A/A′ region of U3 snoRNA as probed by reactivity of CMCT modification. (A) Representative data for CMCT modification for 80 nM U3 (box A/A′ + hinge) incubated in the absence or presence of 1.5 μM Imp3 for 45 min and then treated with 20 mg/mL CMCT for 30 min at RT. Sites modified by CMCT were detected by pauses in primer extension and mapped using a sequencing ladder. Lane intensity quantification is shown on the right for lane 1 (dashed black), lane 2 (red), lane 3 (cyan), and lane 4 (green); each lane was normalized based on counts from a band near the primer (band not shown) that remained unchanged in each lane. Lane T, here and in Figures 5A and 6A, is the dideoxy TTP sequencing ladder. (B) Bases that are protected (open circles) or enhanced (filled circles) upon addition of Imp3 (red), Imp4 (cyan), and both proteins (green) as compared to lane 1 (A) are mapped on the secondary structure of the U3 box A/A′ structure. Parentheses indicate small effects at 32 and 35.