GTP Hydrolysis by IF2 Guides Progression of the Ribosome into Elongation  R. Andrew Marshall, Colin Echeverría Aitken, Joseph D. Puglisi  Molecular Cell  Volume 35, Issue 1, Pages 37-47 (July 2009) DOI: 10.1016/j.molcel.2009.06.008 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Intersubunit FRET between Specifically Labeled Ribosomal Subunits Reports on Intersubunit Dynamics without Interfering with Ribosome Function (A) Surface representation of the E. coli 70S ribosome, showing the location of Cy3 (green) and Cy5 (red) labels. The 30S subunit (gray) is labeled at helix 44, whereas the 50S subunit (blue) is labeled at helix 101. (B) View of the 30S (gray) and 50S (blue) subunits from the subunit interface, with an overlay of the estimated binding footprint for the initiation factors (IFs), modeled from structural studies. (C) Surface immobilization of Cy3-labeled 30S preinitiation complexes containing 30S subunits (gray), initiation factors (green), tRNA, and mRNA followed by delivery of Cy5-labeled 50S (blue) results in formation of a 70S initiation complex and establishment of a FRET signal sensitive to intersubunit conformation. (D and E) Representative fluorescence versus time trajectories obtained from single 70S complexes. Raw fluorescence from Cy3 (green) and Cy5 (red) are used to calculate FRET (blue). Upon stop-flow delivery of cy5-50S, an initial dwell time is observed followed by a burst of FRET. This FRET signal is stable and often censored by the end of observation (D) or less frequently by photophysical events (E). Molecular Cell 2009 35, 37-47DOI: (10.1016/j.molcel.2009.06.008) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Initiation Factors Guide the Assembly of Elongation-Competent 70S Complexes (A and B) FRET intensity histograms for 70S complexes formed in the absence (A) and presence (B) of initiation factors. In the absence of initiation factors, 70S complexes form equally in both the rotated (green curve, low-FRET) and nonrotated (red curve, high-FRET) conformations, whereas initiation factors preferentially select the nonrotated (high-FRET) conformation. Molecular Cell 2009 35, 37-47DOI: (10.1016/j.molcel.2009.06.008) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 IF2-Catalyzed GTP Hydrolysis Drives Subunit Rotation and Commits the Ribosome to Elongation (A) FRET intensity histograms comparing 70S complexes formed in the presence of GTP (blue) with 70S complexes formed in the presence of GDPNP (red). (B–D) Two-dimensional FRET histograms postsynchronized to the arrival of FRET reveal a transient low-FRET state in the progression to high FRET in the presence of GTP (C), whereas 70S complexes formed in the presence of GDPNP (B) proceed directly to low FRET. Magnification of the GTP histogram (D) shows a low-FRET state with an approximate lifetime of 20–30 ms. Molecular Cell 2009 35, 37-47DOI: (10.1016/j.molcel.2009.06.008) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 A Model for Translation Initiation, Highlighting the Role of IF2 mRNA, tRNA, and the initiation factors assemble on the 30S subunit (gray) to form the 30S preinitiation complex, followed by 50S arrival to form the 70S initiation complex. The ribosomal subunits are initially assembled in the rotated conformation, with the peptidyl stem of the initiator tRNA held near the E site by IF2 (yellow). GTP hydrolysis by IF2 occurs rapidly, driving the alignment of the subunits into the nonrotated conformation and moving the peptidyl stem of initiator tRNA into the P site in preparation for peptidyl transfer. Following dissociation of the initiation factors, the nonrotated 70S is committed to enter the elongation cycle. Molecular Cell 2009 35, 37-47DOI: (10.1016/j.molcel.2009.06.008) Copyright © 2009 Elsevier Inc. Terms and Conditions