Detecting protein-induced folding of the U4 snRNA kink-turn by single-molecule multiparameter FRET measurements.

The kink-turn (k-turn), a new RNA structural motif found in the spliceosome and the ribosome, serves as a specific protein recognition element and as a structural building block. While the structure of the spliceosomal U4 snRNA k-turn/15.5K complex is known from a crystal structure, it is unclear whether the k-turn also exists in this folded conformation in the free U4 snRNA. Thus, we investigated the U4 snRNA k-turn by single-molecule FRET measurements in the absence and presence of the 15.5K protein and its dependence on the Na(+) and Mg(2+) ion concentration. We show that the unfolded U4 snRNA k-turn introduces a kink of 85 degrees +/- 15 degrees in an RNA double helix. While Na(+) and Mg(2+) ions induce this more open conformation of the k-turn, binding of the 15.5K protein was found to induce the tightly kinked conformation in the RNA that increases the kink to 52 degrees +/- 15 degrees . By comparison of the measured FRET distances with a computer-modeled structure, we show that this strong kink is due to the k-turn motif adopting its folded conformation. Thus, in the free U4 snRNA, the k-turn exists only in an unfolded conformation, and its folding is induced by binding of the 15.5K protein.