Journal of Molecular Biology
Volume 373, Issue 4, 2 November 2007, Pages 1058-1070
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Analysis of Structural Dynamics in the Ribosome by TLS Crystallographic Refinement

https://doi.org/10.1016/j.jmb.2007.08.054Get rights and content

Abstract

A major goal in the study of ribosome structure and function is to obtain a complete description of the conformational dynamics of the ribosome during the many steps of protein synthesis. Here, we report a new approach to the study of ribosome dynamics using translation-libration-screw (TLS) refinement against experimental X-ray diffraction data. TLS analysis of complexes of the 70 S ribosome suggests that many of its structural features have an inherent tendency for anisotropic movement. Analysis of displacements of the 30 S and 50 S ribosomal subunits reveals an intrinsic bias for “ratchet-like” intersubunit rotation. The libration axes for both subunits pass through the peptidyl transferase center (PTC), indicating a tendency for structural rotations to occur around the site of peptide bond formation. The modes of anisotropic movement of ribosomal RNA components, including the head of the 30 S subunit, the L1 and L11 stalks and the two main arms of the tRNAs were found to correlate with their respective modes of movement previously inferred from comparisons of ribosomes trapped in different functional states. In the small subunit, the mobilities of features interacting with the Shine–Dalgarno helix are decreased in the presence of the Shine–Dalgarno helix, supporting the proposal that that formation of the Shine–Dalgarno helix during initiation may contribute to stabilization of the small subunit for optimal interaction with initiator tRNAfMet. The similarity of TLS parameters for two independently solved structures of similar ribosome complexes suggests that TLS analysis can provide useful information about the dynamics of very large macromolecular objects and at resolutions lower than those at which TLS refinement has commonly been applied.

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.

References (45)

  • A. Korostelev et al.

    Crystal structure of a 70S ribosome-tRNA complex reveals functional interactions and rearrangements

    Cell

    (2006)
  • M. Valle et al.

    Locking and unlocking of ribosomal motions

    Cell

    (2003)
  • H. Gao et al.

    Study of the structural dynamics of the E coli 70S ribosome using real-space refinement

    Cell

    (2003)
  • J. Harms et al.

    High resolution structure of the large ribosomal subunit from a mesophilic eubacterium

    Cell

    (2001)
  • H. Stark et al.

    Large-scale movement of elongation factor G and extensive conformational change of the ribosome during translocation

    Cell

    (2000)
  • A. Dallas et al.

    Interaction of translation initiation factor 3 with the 30S ribosomal subunit

    Mol. Cell

    (2001)
  • L. Lancaster et al.

    Involvement of 16S rRNA nucleotides G1338 and A1339 in discrimination of initiator tRNA

    Mol. Cell

    (2005)
  • J. Frank et al.

    A ratchet-like inter-subunit reorganization of the ribosome during translocation

    Nature

    (2000)
  • L.H. Horan et al.

    Intersubunit movement is required for ribosomal translocation

    Proc. Natl. Acad. Sci. USA

    (2007)
  • K. Fredrick et al.

    Catalysis of ribosomal translocation by sparsomycin

    Science

    (2003)
  • D.W.J. Cruickshank

    The determination of the anisotropic thermal motion of atoms in crystals

    Acta Crystallog.

    (1956)
  • J.D. Dunitz et al.

    Interpretation of atomic displacement parameters from diffraction studies of crystals

    J. Phys. Chem.

    (1988)
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