Fig. 4.
The stable box A/A’ stem structure of U3 MINI is unfolded to U3 MINI* by addition of protein, primarily Imp3p. (a) OD260 values for unlabeled U3 MINI melted in the forward (circles) and refolded in the reverse direction (squares). A smoothed derivative plot (dashed line) of the forward melt indicates a Tm of 54 °C, with an enthalpy of 39 kcal/mol and a ΔG°20 °C of 4 kcal/mol. (b) To monitor distance changes at the base of the box A/A’ stem structure trFRET was performed using a doubly labeled substrate: Fl is attached to the 5’ end of U3 MINI via a six carbon linker and an internal Rh label is attached via a longer succinimide linker to C5 of uracil 38. (c) FRET distance distributions between the donor and acceptor of Fl-U3 MINI-Rh upon binding of Imp3p, Imp4p, and 18S as determined by trFRET (Supplemental Fig. S3 contains decay curves). In the absence of protein ~93% fraction of molecules show a distance distribution centered around 19 Å (grey line), with a full width at half-maximum (fwhm) of 18 Å. The fwhm reflects in part the intrinsic flexibility of the RNA in solution. A smaller ~7% fraction (dashed grey line) has distance distribution centered around 45 Å with an fwhm of 37 Å and is likely to result from a small population of U3 MINI dimer. Using an electrophoretic mobility shift assay, the inset shows that 4 ± 2 % of U3 MINI exists in a dimer at the 0.5 µM concentration used for the trFRET studies. Upon binding of Imp3p (dashed black line), a single distance distribution is obtained, centered at a distance of ~32 Å with a fwhm of 8 Å. Subsequent binding of Imp4p (dotted black line) and 18S (solid black line) shows no significant additional change in distance distribution (mean distance of 33 Å with an fwhm of 9 Å and mean distance of 34 Å with an fwhm of 10 Å, respectively). (d) The simplest model indicates that the unfolded RNA nucleotides loop back to permit the separation distance to be independent of Imp4p and 18S binding. Schematics illustrate chaperone complex unfolding the box A/A’ stem of U3 MINI from an A-form helix with a FRET pair separated by 19 Å (left panel) to U3 MINI* where the separation increases to ~32 Å upon binding of Im3p3 and Imp4p (middle panel). Addition of 18S results in a negligible increase in the separation of the FRET pair (right panel).