Molecular dynamics simulations suggest that RNA three-way junctions can act as flexible RNA structural elements in the ribosome

Published online 27 May 2010 Nucleic Acids Research, 2010, Vol. 38, No. 18 6247–6264 doi:10.1093/nar/gkq414 Molecular dynamics simulations suggest that RNA three-way junctions can act as flexible RNA structural elements in the ribosome Ivana Beššeová1,2, Kamila Réblová1, Neocles B. Leontis3,* and Jiřı́ Šponer1,* 1 Institute of Biophysics, Academy of Sciences of the Czech Republic, 61265 Brno, 2Gilead Sciences & IOCB Research Center, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 166 10 Prague 6, Czech Republic and 3Department of Chemistry, Bowling Green State University, Bowling Green, OH 43403, USA Received January 29, 2010; Revised May 3, 2010; Accepted May 4, 2010 ABSTRACT We present extensive explicit solvent molecular dynamics analysis of three RNA three-way junctions (3WJs) from the large ribosomal subunit: the 3WJ formed by Helices 90–92 (H90–H92) of 23S rRNA; the 3WJ formed by H42–H44 organizing the GTPase associated center (GAC) of 23S rRNA; and the 3WJ of 5S rRNA. H92 near the peptidyl transferase center binds the 30 -CCA end of amino-acylated tRNA. The GAC binds protein factors and stimulates GTP hydrolysis driving protein synthesis. The 5S rRNA binds the central protuberance and A-site finger (ASF) involved in bridges with the 30S subunit. The simulations reveal that all three 3WJs possess significant anisotropic hinge-like flexibility between their stacked stems and dynamics within the compact regions of their adjacent stems. The A-site 3WJ dynamics may facilitate accommodation of tRNA, while the 5S 3WJ flexibility appears to be essential for coordinated movements of ASF and 5S rRNA. The GAC 3WJ may support large-scale dynamics of the L7/L12-stalk region. The simulations reveal that H42–H44 rRNA segments are not fully relaxed and in the X-ray structures they are bent towards the large subunit. The bending may be related to L10 binding and is distributed between the 3WJ and the H42–H97 contact. INTRODUCTION RNA three-way junctions (3WJs) are common elements of structured RNAs that result from the base pairing of three distinct strand segments of the RNA sequence so as to form three helices diverging from a single point in the secondary structure. 3WJs consist of six components: Three helices (designated P1, P2 and P3) and three single-stranded joiner segments (J12, J23 and J31) (1). In most structured 3WJs, two of the helices stack coaxially (Figure 1A). Based on the coaxial stacking patterns and the lengths of the linker segments, 3WJs have been classified into three basic types, A, B and C (1). In type C 3WJs, which are the most common, J31 is longer than J23 and helices P1 and P2 are coaxially stacked. In addition, P1 and P2 are usually directly connected (i.e. J12 has length 0) and P3 is tilted towards P1, close enough for tertiary interactions to form between P1 and P3, some distance away from the junction. The J31 segment generally forms a loop-like structure interacting with the minor groove of P2. In several instances, J31 forms a small hairpin-like RNA motif closed by a single base pair. We complement structural studies of RNA 3WJs with explicit solvent molecular dynamics (MD) simulations. While MD is limited by a number of approximations, especially simplifying assumptions of the force field (2–7), it can nonetheless capture the basic intrinsic flexibility of RNA modular motifs and thus help to interpret experimental data (8–17). MD can provide insights into dynamical features of RNA that are not fully apparent from structural studies that typically reveal static averaged structures. Large RNA-based nanomachines like the ribosome work in the regime of high viscosity and very low inertia so that the essential principles of their function are strikingly different from those of macroscopic machines (18–24). They use chemical energy (in the form of GTP) to rectify random thermal fluctuations into directional motion. Thus, during the protein synthesis cycle, each cognate tRNA is transported (along with the bound mRNA) directionally across the interface between the large and small subunits of the ribosome, from the *To whom correspondence should be addressed. Tel: +420 541517133; Fax: +420 541212179; Email: Correspondence may also be addressed to Neocles B. Leontis. Tel: 419 372 8663; Fax: 419 372 9809; Email: ß The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 6248 Nucleic Acids Research, 2010, Vol. 38, No. 18 Figure 1. (A) Schematic representation of the components of RNA 3WJs. The P1/P3 contact occurs only in type C 3WJs. Simulated systems: (B) H90–H92 segment of 23S rRNA; (C) H42–H44 segment of 23S rRNA; and (D) H1–H3 and H5 of 5S rRNA. The junction regions are shown in color as in (A). The gray regions in (B) and (C) are included in the simulations, but are formally outside the junction. Black lines (AB, PP1–PP4 and EF) represent the distances describing the motions (see below). Tertiary A-minor I and A-minor II interactions (57,58) (or equivalent interactions) of the junction region (see below) are shown in gray and black licorice representations. A-site to the P-site and to the E-site. The subunits and their flexible parts also move relative to each other in a coordinated and cyclical manner. These motions are largely driven by stochastic processes, where fluctuations are of utmost importance. MD can shed light on the overall stochastic and anisotropic flexibilities of the RNA building blocks that are important for their function and which usually are not apparent from the structural studies. Simulations can further provide atomistic insights into the structural dynamics (2,4,7,9– 11,14,17,25–27). MD captures motions that occur on the picosecond to sub-microsecond timescale and are therefore subject to low energy barriers. Due to the simulation timescale, simulations of RNA structural modules extracted from larger structures (e.g. 3WJs extracted from the ribosome) show dynamics pertinent to the starting functional geometry of the studied RNA. This geometry may be different from non-functional structures formed in equilibrium solution experiments (28). The fluctuations seen in simulations characterize the intrinsic flexibility of the studied RNA building blocks, which can be used to achieve functional movements. Due to limitations such as uncertainties in the starting structures (which are generally medium resolution, at best) and approximations inherent to the force fields, the simulation results should not be overinterpreted. For the purpose of basic understanding of RNA flexibility, however, the method is robust. We investigate the basic dynamical properties and flexibility of repr (...truncated)