Volume 44, Issue 2, Pages 214-224 (October 2011) Structure and Dynamics of the Mammalian Ribosomal Pretranslocation Complex  Tatyana Budkevich, Jan Giesebrecht, Roger B. Altman, James B. Munro, Thorsten Mielke, Knud H. Nierhaus, Scott C. Blanchard, Christian M.T. Spahn  Molecular Cell  Volume 44, Issue 2, Pages 214-224 (October 2011) DOI: 10.1016/j.molcel.2011.07.040 Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 Multiparticle Refinement Reveals Four Structurally Distinct Subpopulations in the PRE Complex (A, B) Representation of the two nonrotated populations with three classically positioned A-, P-, and E-site tRNAs. (C and D) Representation of the two rotated substates, where deacylated tRNA occupies a P/E hybrid state while the peptidyl-tRNA body adopts configurations that are similar to classical (C) or hybrid (D) positions. Color code: 60S subunit (blue), 40S subunit (yellow), A-site tRNA (pink), P-site tRNA (green), E-site tRNA (orange). See also Figure S1 (Heterogeneity in the 80S Ribosome PRE Complexes from Rabbit Liver) and Figure S2 (Resolution Curves for the Four Subpopulations of the Mammalian PRE Complex). Molecular Cell 2011 44, 214-224DOI: (10.1016/j.molcel.2011.07.040) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Analysis of Classically Positioned tRNAs Observed in the Classical-1 PRE Complex from Rabbit Liver Deviation in the classical tRNA positions near the elbow regions of tRNA observed in the nonrotated 80S PRE complex populations (colored ribbon) compared to those observed in T.thermophilus 70S structures after aligning the small subunits (Voorhees et al., 2009) (gray ribbon, PDB ID 2WDG). Distances were measured between the phosphate backbone at position 56. Color code: A-site tRNA (pink), P-site tRNA (green), E-site tRNA (orange). See also Figure S3 (tRNA in the Classical A/A, P/P and E/E Positions), Table S1 (tRNA-40S Contacts), and Table S2 (tRNA-60S Contacts). Molecular Cell 2011 44, 214-224DOI: (10.1016/j.molcel.2011.07.040) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 Eukaryotic-Specific Contacts between tRNAs and 80S Ribosomes Detected within the Classical-1 80S PRE Complex (A) Contact of the A-site tRNA acceptor stem with H89 of the 60S ribosomal subunit. (B) Contact of the A-site tRNA anticodon stem (positions 28/29) with the 40S ribosomal subunit. (C) Contacts between the T-loop of P-site tRNA and elements (H85, H82/83, and/or rpL44e) of the 60S ribosomal subunit. The models for rRNA helices and ribosomal proteins are derived from X-ray structures of the yeast 80S ribosome (Ben-Shem et al., 2010) for the 60S subunit (PDB ID 3O58) and the 40S-eIF1 complex (Rabl et al., 2011) for the 40S subunit (PDB ID 2XZN). Molecular Cell 2011 44, 214-224DOI: (10.1016/j.molcel.2011.07.040) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Comparison of A-Site tRNA Contacts with 60S Ribosomal Subunit for All Four Subpopulations Described (A–H) From top to bottom: A, B, classical-1; C, D, classical-2; E, F, rotated-1; G, H rotated-2. (A, C, E, and G) Illustration of different contacts between A-site tRNA and H69. (B, D, F, and H) Demonstration of the potentially dynamic interaction between the A-site tRNA elbow and components of the 60S subunit during the transition from classical-1 to rotated-2 intermediate states. Models for rRNA and separated proteins are derived from the X-ray structure of the yeast 80S ribosome (Ben-Shem et al., 2010) (PDB ID 3O58). See also Figure S4, (Distinct Features of 80S Ribosomes in the Classical-1 and Classical-2 Subpopulations), Movie S2 (Spontaneous Movement of tRNAs Relative to the 60S Subunit), and Movie S3 (Spontaneous Movement of tRNAs Relative to the 40S Subunit). Molecular Cell 2011 44, 214-224DOI: (10.1016/j.molcel.2011.07.040) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 Comparison of tRNA Positions in Rotated-1 and Rotated-2 Subpopulations of 80S PRE Complex (A) Superposition of 40S subunits separated from classical-1 (gray mesh) and rotated-2 (yellow solid) subpopulations tracks A/A and P/P tRNAs movements inside the intersubunit cavity (inset shows the same scene with usage of crystallographic models derived from Voorhees et al. [2009]), PDB ID 2WDG). (B) Superposition of ASL regions of classical (A/A) tRNA (gray mesh and X-ray model) with hybrid (A/P) tRNA (pink, map and model derived from Ratje et al. 2010, PDB ID 2XSY.pdb) indicates a conformational difference between the two structures. A similar distortion (kink) is even more pronounced for P/P (gray mesh, X-ray model) and P/E (green solid map and model PDB ID 2XSY.pdb) tRNAs. See also Figure S5 (Rotation of the 40S Subunit in the Nonrotated and Rotated Subpopulations). Molecular Cell 2011 44, 214-224DOI: (10.1016/j.molcel.2011.07.040) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 tRNA Dynamics on the Mammalian PRE Complex Bearing Fluorescently Labeled tRNAs in the A and P Sites (A) 80S PRE complexes containing deacylated Cy3-labeled tRNAPhe in the P site and Cy5-labeled NAc-Phe-Lys-tRNALys in the A site prepared as described in Experimental Procedures. (B) 80S PRE complexes imaged in the presence of 5 μM deacylated E.coli tRNAfMet. (C) 80S PRE complexes imaged in the presence of 200 μM cycloheximide. For each panel, the nature of the complex investigated and the putative motions of tRNA between classical and hybrid positions is schematized (left panel) along with population histograms showing the distribution of FRET values observed (center panel), as well as representative single-molecule FRET trajectories for individual molecules from within each experiment overlaid by the idealization (red) obtained through hidden Markov modeling (Experimental Procedures). See also Figure S6 (Inclusion of Deacylated tRNA in the Imaging Buffer Increased E-site tRNA Occupancy in a Concentration-Dependent Fashion). Molecular Cell 2011 44, 214-224DOI: (10.1016/j.molcel.2011.07.040) Copyright © 2011 Elsevier Inc. Terms and Conditions