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

Abstract

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 Mg²⁺ ion concentration. We show that the unfolded U4 snRNA k-turn introduces a kink of 85° ± 15° in an RNA double helix. While Na⁺ and Mg²⁺ 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° ± 15°. 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.

Keywords

• U4 snRNA k-turn
• 15.5K
• protein-induced folding
• multiparameter fluorescence detection
• fluorescence resonance energy transfer
• FRET