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 Mg 2+ 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 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°-15°. By comparison of the measured FRET distances with a computer-modeled structure, we show that this strong kink is due to the kturn 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.

Kink turn sRNA folding upon L7Ae binding using molecular dynamics simulations

“…32,35,40 To quantitatively identify this motion, principal component analysis (PCA) was carried out on the bound sRNA separately for each trajectory. In general, the three most principal components (named PC1, PC2, and PC3) represent over 80% of the overall fluctuations for ten trajectories.…”

The role of specific 2′-hydroxyl groups in the stabilization of the folded conformation of kink-turn RNA

“…The structural principles of K-turns have been analyzed in detail using isostericity matrices by Westhof and colleagues (Lescoute et al 2005). 1 The structure of free K-turn RNA is very dynamic and dependent on the binding of metal ions (Goody et al 2003) or L7Ae-related protein (Turner et al 2005;Wozniak et al 2005). In the absence of protein binding, K-turn RNA exists in an equilibrium between the tightly kinked and a more extended structure, and the position of equilibrium is determined by metal ion concentration.…”

A structural database for k-turn motifs in RNA

“…They are very commonly found in C/D and H/ACA guide snoRNAs and in U3 snoRNA species (Kuhn et al 2002;Watkins et al 2002;Bortolin et al 2003;Marmier-Gourrier et al 2003;Rozhdestvensky et al 2003;Hamma and Ferré-D'Amaré 2004;Moore et al 2004;Szewczak et al 2005;Youssef et al 2007). There is also a near-canonical k-turn in a stem-loop of the human U4 snRNA (Vidovic et al 2000;Wozniak et al 2005). Some k-turns have been identified unequivocally, from their structure in situ within crystal structures of larger RNA structures or complexes.…”