Journal of Molecular Biology
Analysis of Structural Dynamics in the Ribosome by TLS Crystallographic Refinement
Introduction
Ribosomes are the ribonucleoprotein particles responsible for converting genetic information encoded in messenger RNA (mRNA) into proteins. During protein synthesis, transfer RNAs (tRNAs) deliver amino acids to the ribosome. Recognition of the cognate aminoacyl-tRNA for each mRNA codon (decoding process) occurs on the small (30 S) subunit and is followed by peptide bond formation in the peptidyl-transferase center (PTC) of the large subunit. Each cycle of elongation is followed by translocation of tRNAs through the ribosome, during which tRNA moves from the A (aminoacyl) site to the P (peptidyl) site and then to the E (exit) site before leaving the ribosome. These functional processes are accompanied by rapid, large-scale molecular movements of tRNA, mRNA and translation factors that are believed to be accompanied by corresponding movements in the structure of the ribosome. For example, comparison of cryo-electron microscopic reconstructions of ribosomes trapped in different states of translocation has led to a model for translocation based on intersubunit rotation1 that has recently been supported by solution studies using FRET2 and intersubunit cross-linking.3 Although translocation normally requires participation of elongation factor EF-G and GTP, it has been shown to occur in their absence under certain in vitro conditions,4., 5., 6., 7., 8. indicating that the ribosome has an inherent ability to undergo the conformational rearrangements required for translocation. It seems likely that the structure of the ribosome has been optimized during evolution to support these and other dynamic events that underlie protein synthesis. A major goal in the study of ribosome structure and function is to obtain a full description of the molecular dynamics of translation, in terms of the structures of the ribosome and its functional ligands. The studies presented here suggest a novel approach toward this end.
Since the magnitudes and directionalities of atomic displacements are captured in diffraction data,9 these data can be exploited to infer information about macromolecular dynamics. Attributing individual atomic anisotropic displacement parameters to a model, however, is possible only when high-resolution (better than 1.2 Å) diffraction data are available.10 However, at lower resolutions (commonly 1.2 to 3 Å),11 translation-libration-screw (TLS) formalism can be applied to rigid domains, rather than to individual atoms, and has been demonstrated to closely approximate the anisotropic behavior of an atomic model.12., 13. Comparison of TLS parameters with the amplitudes of normal modes describing the internal motion of a protein indicates that TLS parameters for the overall motion make the largest contribution to atomic displacements.14., 15. TLS analysis has been shown to provide biologically relevant information, such as identification of mobile domains contributing to induced fit of a protein as well as of regions of restricted mobility, comprised of biologically critical sites, such as the active sites of enzymes.16., 17. The theory underlying TLS parameterization has been presented in detail by Schomaker and Trueblood18., 19. and Howlin et al.20 In summary, apart from improving agreement between an atomic structure and diffraction data by approximating anisotropic disorder of atoms, TLS refinement yields translational, librational and screw tensors for the respective rigid groups. The collective anisotropic disorder of atoms representing each rigid group can therefore be described as translation along translational axes, torsional oscillations around librational axes and screw motions along screw axes. In this work, TLS formalism has allowed visualization of the anisotropic dynamics of the ribosomal subunits and their sub-structures, and tRNAs bound to the ribosome.
Section snippets
TLS refinement
The structure used for TLS analysis represents an elongation-like ribosomal complex containing a short ten-nucleotide defined mRNA, tRNAPhe bound to the P site and an endogenous mixture of tRNAs bound to the E site (modeled as tRNAPhe), determined at 3.7 Å resolution,21 referred to below as the tRNAPhe complex. TLS refinement followed a conventional refinement strategy and led to reduction in crystallographic R and free R factors, indicating improvement of the fit of the structural model to the
Conclusions
The observed atomic displacements in the 70 S ribosome complex are not random in nature but are directed along pathways that often coincide with the directions of movements that are believed to accompany the processes of protein synthesis. The magnitudes of the TLS librations, however, do not always strictly reflect the functional dynamics of the ribosome, but are sometimes also influenced by constraints dictated by the crystallographic environment. For the most part, the observed anisotropic
Starting models and diffraction data
The structures of the 3.7 Å tRNAPhe21 and 3.8 Å tRNAfMet22 from Thurmus thermophilus ribosomal complexes containing mRNA, P-and E-tRNAs were downloaded from the Protein Data Bank40 (PDB accession codes 2OW8 and 1VSA for the former and 2QNH for the latter). Their structures were obtained by real-space41 and reciprocal-space simulated annealing torsion-angle dynamics and B-group refinement methods42 as described in Korostelev et al.,21 with the starting models for the structures of ribosomal
Acknowledgements
This work was supported by grants no. GM-17129 and GM-59140 (to H.F.N.) from the NIH. We thank Sergei Trakhanov and Martin Laurberg for their contributions to structure determination.
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