Relating Structure and Dynamics in RNA Biology.

Relating Structure and Dynamics in RNA Biology Kevin P. Larsen,1,2,4 Junhong Choi,1,3,4 Arjun Prabhakar,1,2 Elisabetta Viani Puglisi,1 and Joseph D. Puglisi1 1 Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305 2 Biophysics Program, Stanford University, Stanford, California 94305 3 Department of Applied Physics, Stanford University, Stanford, California 94305 Correspondence: ; SUMMARY Recent advances in structural biology methods have enabled a surge in the number of RNA and RNA–protein assembly structures available at atomic or near-atomic resolution. These complexes are often trapped in discrete conformational states that exist along a mechanistic pathway. Single-molecule fluorescence methods provide temporal resolution to elucidate the dynamic mechanisms of processes involving complex RNA and RNA–protein assemblies, but interpretation of such data often requires previous structural knowledge. Here we highlight how single-molecule tools can directly complement structural approaches for two processes–– translation and reverse transcription—to provide a dynamic view of molecular function. Outline 1 Introduction 2 Structural dynamics of the ribosome during decoding 3 Modulation of translation decoding dynamics by m6A mRNA modifications 4 Modulation of translation decoding dynamics by 2′ -O-methylation 5 Next steps in studying the role of mRNA modification in translation decoding 6 Structural dynamics of HIV-1 reverse transcription initiation 7 HIV-1 viral RNA–tRNALys 3 complex structure and heterogeneity 8 Interactions of HIV-1 RT with the primer–template complex 9 Insights into the tertiary structure of the HIV-1 RT initiation complex 10 Next steps in studying HIV-1 reverse transcription initiation 11 Concluding remarks References 4 These authors contributed equally to this work. Editors: Thomas R. Cech, Joan A. Steitz, and John F. Atkins Additional Perspectives on RNA Worlds available at www.cshperspectives.org Copyright # 2019 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a032474 Cite this article as Cold Spring Harb Perspect Biol 2019;11:a032474 1 K.P. Larsen et al. 1 INTRODUCTION macromolecular conformation and in chemical composition (ligands or factors bound) and may proceed along different mechanistic pathways. Single-molecule methods have provided a bridge between structure and dynamics, allowing real-time analysis of RNAs and their assemblies. Here we address the interplay of structure and dynamics, using examples from our recent work on translation by the ribosome and HIV reverse transcription. The three major structural methods—X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM)—provide different portals into dynamics (Fig. 1). X-ray crystallography The past decades have seen an explosion in structures of RNA and RNA–protein assemblies, including the ribosome and spliceosome. These structures have revealed the basic molecular principles of RNA secondary and tertiary folding and recognition of RNAs by structured and unstructured protein domains. Yet these structures represent static snapshots of the dynamic RNA–protein machinery of the cell. What has been lacking are time-resolved methods to link detailed structural views into a mechanistic movie of biological systems. These systems evolve temporally in both NMR A C–G A–U C–G + Paro. C–G G U U C Freezing on EM grids 1H (ppm) UOU C–G AOA Cryo-EM Hetergeneous RNP sample 1H (ppm) A-site rRNA alone 1H (ppm) 5′ 3′ G–C G–C C–G G–C – Paro. Classification analysis 1H (ppm) A-site rRNA with Paro bound A-site rRNA helix Different structures resolved X-ray crystallography E P Single-molecule FRET A RNP complex (Ribosome) Immobilization and imaging Crystallization RNP sample RNP structure 1 0.8 0.6 0.4 0.2 0 –0.2 Postanalysis –2 4 0 2 Time (sec) 6 FRET efficiency FRET efficiency modeling FRET efficiency calculation Postsynchronized structural change X-ray diffraction Time Figure 1. Major structural methods to study RNA–RNA–protein complexes. (Top left) Structures of ribosomal RNA (rRNA) helix with or without antibiotic paromomycin (Paro). (Adapted from Fourmy et al. 1998.) (Bottom left) High-resolution structure of ribosome using X-ray crystallography. (Top right) Cryo-electron microscopy (cryo-EM) structures of ribosome, with or without A-site transfer RNA (A-tRNA). (Bottom right) Single-molecule Förster resonance energy transfer (FRET) data showing binding of A-tRNA to the ribosome. (Data adapted from Fourmy et al. 1998 for nuclear magnetic resonance [NMR] spectroscopy [Protein Data Bank (PDB) IDs 1FYP, 1FYO], PDB ID 4V51 for X-ray crystallography, 5JTE for cryo-EM, and Choi et al. 2018 for single-molecule FRET.) 2 Cite this article as Cold Spring Harb Perspect Biol 2019;11:a032474 Relating Structure and Dynamics in RNA Biology requires crystallization of macromolecular complexes into regular lattices, which in certain cases can skew conformations because of intermolecular packing interactions. Data in X-ray diffraction are derived from coherent scattering of the X-ray beam by all members of the crystalline lattice. Dynamic information is often inferred from so-called B-factors, or “temperature factors,” that reflect uncertainty in the location of atoms—high B-factors can result from dynamic regions in a macromolecule or from errors during modeling. Time-resolved conformational changes have been observed but require triggering of all members of the lattice simultaneously. Synchronizing conformational changes within the crystalline lattice often requires an external cue, typically light, to initiate a reaction (Srajer et al. 1996; Jung et al. 2013). Recent advances in the field of coherent X-ray sources (X-ray free-electron lasers or XFELs) have enabled faster data acquisition on nanocrystalline materials (Chapman et al. 2011; Tenboer et al. 2014). The fast data acquisition at room temperature is coupled with small crystal size to allow rapid mixing with ligands that can enter the crystal lattice. This has led to X-ray studies on conformational changes induced by ligands that bind to RNA (Stagno et al. 2017). Although still in its infancy and often restricted to specific cases, X-ray crystallography using XFELs shows potential for studying complex biomolecular dynamics. NMR spectroscopy is a powerful probe of chemical dynamics, over a broad range of timescales and processes. NMR relaxation measurements can probe residue-byresidue motions on timescales of 10−9 to 10−5 sec to reveal fast movements within biomolecules. Advances in using relaxation dispersion methods have allowed investigation of “excited,” lowly populated states for RNAs and proteins at intermediate timescales; the work of Al-Hashimi and coworkers in particular has shown how RNA can sample alternate pairings on a micro to millisecond range (AlHashimi 2013). NMR (...truncated)