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

Ivana Bes s eova 1 2 Kamila R eblova 2 Neocles B. Leontis 0 Jir S poner 2 0 Department of Chemistry, Bowling Green State University , Bowling Green, OH 43403, USA 1 Gilead Sciences & IOCB Research Center, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic , 166 10 Prague 6, Czech Republic 2 Institute of Biophysics, Academy of Sciences of the Czech Republic , 61265 Brno 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. - 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 (27), it can nonetheless capture the basic intrinsic flexibility of RNA modular motifs and thus help to interpret experimental data (817). 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 (1824). 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 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 11,14,17,2527). 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 representative RNA 3WJs, specifically type C 3WJ (Figures 1 and 2). First, we studied a 76-nt segment containing the 3WJ that comprises helices 90, 91 and 92 (H90, H91 and H92) of 23S rRNA and is located adjacent to the peptidyl transferase center (PTC). H90 corresponds to P1, H91 to P2 and H92 to P3. We call this 3WJ the A-site junction because the hairpin loop of H92, the so-called A-loop of 23S rRNA, binds the 30-CCA end of A-site aminoacyl-tRNA (aa-tRNA). H90 is one of the helices composing the multi-helix junction that has been identified as the PTC of 23S rRNA. H90 covalently anchors the A-site junction to the PTC, while H91 stacks coaxially with H90 and projects away from the PTC toward the upper surface of the 50S subunit in the standard view, in which the L1-site, the central protuberance (CP) and the GTPase associated center (GAC) all project upwards (Figure 2). H91 provides a docking site for the short hairpin loop formed by the nucleotides that connect H92 to H90, which corresponds to J31 in Figure 1A. This hairpin loop stacks on H92 and comprises three stacked adenosines (nucleotides 25642566 in the Escherichia coli numbering). The conserved bases A2566 and U2562 form a trans H/WC base pair (29) that closes the loop. This short element has also been called H92a. H91 forms several other tertiary interactions, in addition to stacking on H90 and providing a docking site for H92a (J31). The A-site junction provides a potentially flexible point of connection for H92. The A-site loop of H92 helps to position the aminoacyl group in the PTC during the protein synthesis. This is i (...truncated)