The kink-turn motif in RNA is dimorphic, and metal ion-dependent

Abstract

The kink-turn (K-turn) is a new motif in RNA structure that was identified by examination of the crystal structures of the ribosome. We examined the structural and dynamic properties of this element in free solution. The K-turn RNA exists in a dynamic equilibrium between a tightly kinked conformation and a more open structure similar to a simple bulge bend. The highly kinked form is stabilized by the noncooperative binding of metal ions, but a significant population of the less-kinked form is present even in the presence of relatively high concentrations of divalent metal ions. The conformation of the tightly kinked population is in excellent agreement with that of the K-turn structures observed in the ribosome by crystallography. The end-to-end FRET efficiency of this species agrees closely with that of the ribosomal K-turn, and the direction of the bend measured by comparative gel electrophoresis also corresponds very well. These results show that the tightly kinked conformation of the K-turn requires stabilization by other factors, possibly by protein binding, for example. The K-turn is therefore unlikely to be of itself a primary organizing feature in RNA.

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

“…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. The kinked conformation confers strongly retarded electrophoretic mobility to the RNA, whereas the extended structure migrates with a mobility typical of a simple 3-nt bulge (Goody et al 2003;Matsumura et al 2003). Fluorescence resonance energy transfer (FRET) measurements (Goody et al 2003) show that the RNA undergoes an ion-induced two-state transition between the two structures, consistent with molecular dynamics studies that have predicted structural bistability in K-turn RNA (Cojocaru et al 2005;Razga et al 2005Razga et al , 2006.…”

“…We have previously shown that RNA duplexes containing a central K-turn sequence exhibit strongly retarded electrophoretic mobility in polyacrylamide in the presence of Mg 2+ ions (Goody et al 2003), consistent with the adoption of a highly kinked conformation. Sequence changes that disrupt the canonical K-turn sequence lead to normal electrophoretic mobility for an RNA duplex of the same length interrupted by a 3-nt bulge.…”

“…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.…”

“…The kinked conformation confers strongly retarded electrophoretic mobility to the RNA, whereas the extended structure migrates with a mobility typical of a simple 3-nt bulge (Goody et al 2003;Matsumura et al 2003). Fluorescence resonance energy transfer (FRET) measurements (Goody et al 2003) show that the RNA undergoes an ion-induced two-state transition between the two structures, consistent with molecular dynamics studies that have predicted structural bistability in K-turn RNA (Cojocaru et al 2005;Razga et al 2005Razga et al , 2006. Single-molecule FRET data indicate that transitions between the kinked and extended forms are fast (B. Turner, J.C. Penedo, and D.M.J.…”

“…The two-state folding process is readily followed by electrophoretic or FRET methods (Goody et al 2003), and there are a number of crystal structures of K-turns in their folded conformation that provide structural insight. In this study we have examined the importance of selected 29-hydroxyl groups in the stabilization of the kinked structure by systematic chemical substitution experiments.…”