The Real-Time Path of Translation Factor IF3 onto and off the Ribosome Attilio Fabbretti, Cynthia L. Pon, Scott P. Hennelly, Walter E. Hill, J. Stephen Lodmell, Claudio O. Gualerzi  Molecular Cell  Volume 25, Issue 2, Pages 285-296 (January 2007) DOI: 10.1016/j.molcel.2006.12.011 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 DMS Probing of the Topographical Localization of IF3 (A) Three-dimensional structure of IF3 (IF3-N + IF3-C domains) (Biou et al., 1995; PDB file 1TIF; Garcia et al., 1995a; PDB file 2IFE), (B) G700 region, and (C) A790 region of 16S rRNA DMS-modified in situ in the presence of WT (native) IF3 or IF3C domain as indicated above the gel and analyzed by primer extension. The bases affected by the factor are indicated by black bars on the right side of the gels, and the bases whose intensities were densitometrically quantified are indicated by the following symbols near the corresponding bars: A704(■□), A706(▾▿), and C708(▴▵) in red and C783(■□), A784(▾▿), A790(▴▵) and A794 (♦⋄) in blue. The individual lanes are indicated: 16S rRNA sequencing (A and G), 16S rRNA without DMS (K), and 30S modified in the absence of factor (30S) or in the presence of the stoichiometric excess of factor (five, ten, and twenty times, as indicated). The intensity variations in the G700 and A790 regions as a function of increasing WT IF3/30S (closed symbols) and IF3C/30S ratios (open symbols) are plotted in the graphs to the right of the corresponding gels. The localization of the modified bases is indicated (D) by arrows in the two-dimensional structure of 16S rRNA and (E) within the three-dimensional structure of the 30S subunit (Vila-Sanjurjo et al., 2003; PDB file 1PNX) according to the following color code: quantified (red) and nonquantified (pink) bases in the G700 region, and quantified (blue) and nonquantified (light blue) bases in the A790 region. Secondary effects occur at bases indicated in green. Molecular Cell 2007 25, 285-296DOI: (10.1016/j.molcel.2006.12.011) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Secondary Effects of IF3 and IF3C Binding on the Exposure of 16S rRNA at the 30S Ribosomal Subunit Surface (A and B) Primer extension analysis of 16S rRNA modified in situ by DMS in the presence of WT (native) IF3 (A) or IF3C (B) was performed as indicated in Experimental Procedures. The affected bases are indicated on the side of the gels. The individual lanes are indicated as in Figures 1B and 1C. (C) Localization in the three-dimensional structure of the 30S subunit of the modified bases (green) with indication of the rRNA helices to which they belong. Ribosomal proteins S11 (blue), S18 (red), and S6 (orange) are also indicated. Molecular Cell 2007 25, 285-296DOI: (10.1016/j.molcel.2006.12.011) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Probing the Topographical Localization of IF3 by In Situ rRNA Cleavage with ONOOK (A and B) Analysis of 16S rRNA protected by increasing amounts of IF3 WT (red) or by IF3C (blue) from in situ ONOOK-induced cleavage in (A) the G700 region and (B) the A790 region. The regions more exposed by the presence of IF3C are indicated in green. The individual lanes are indicated as in Figures 1B and 1C. (C) The regions affected by IF3 or IF3C are indicated by red, blue, or green in the two-dimensional structure of 16S rRNA. Molecular Cell 2007 25, 285-296DOI: (10.1016/j.molcel.2006.12.011) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Validation of the Time-Resolved Probing of 16S rRNA by ONOOK Cleavage (A) Scheme of the experimental design to obtain time-resolved chemical probing with the quench-flow apparatus. (B) Primer extension analysis of a select region (A790) of 16S rRNA probed with ONOOK at 6 mM (left), 12 mM (center), and 18 mM (right) for the times (expressed in milliseconds) indicated above each lane. The “C” lanes contain samples incubated in the absence of ONOOK. (C) the intensity of all resolved bands shown in (B) are plotted as a function of the time allowed for reagent decay (self-quenching) for the reactions carried out with ONOOK at 6 mM (▴), 12 mM (▾), and 18 mM (♦) and without ONOOK (●). Molecular Cell 2007 25, 285-296DOI: (10.1016/j.molcel.2006.12.011) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Time-Resolved Analysis of IF3 Binding to the 30S Ribosomal Subunit Each panel displays the autoradiogram of the primer extension analysis (upper left) and the densitometric quantification of the band intensities (right side), and the three-dimensional structure of 16S rRNA within the 30S subunit (Vila-Sanjurjo et al., 2003; PDB file 1PNX) in the position of the base(s) affected by IF3 is indicated by different colors depending upon their location in the 16S rRNA. Time-resolved chemical probing refers to the following: (A) ONOOK cleavage in the G703 (■)/G688 (▴) region (orange tracings), (B) DMS modification in the A749 (▴)/C750 (■) region (green tracings), (C) ONOOK cleavage in the G700 (red tracings) and A790 (blue tracings) regions, and (D) DMS modification in the G700 (red tracings) and A790 (blue tracings) regions. In (C) and (D), the individual bases are indicated as A704 (▴), A706 (■), C708 (♦), A712 (●), A784 (▵), A790 (○), and A794 (□). In all panels, the reactivity/cleavage of the individual positions is indicated as a function of time elapsed after mixing IF3 with the 30S subunit and is expressed as the percent of the reactivity/cleavage obtained before mixing. The individual lanes are indicated: 16S rRNA sequencing (A and G), 16S rRNA without DMS or ONOOK (K), and 30S modified in the absence of factor (C) or in the presence of IF3 for the indicated times (milliseconds or seconds). All controls and experiments were repeated at least three times, and the standard deviation between the normalized intensity of bands was used to generate error bars. Molecular Cell 2007 25, 285-296DOI: (10.1016/j.molcel.2006.12.011) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Time-Resolved Analysis of the Dissociation of IF3 from the 30S Subunit The upper left side of each panel displays the autoradiogram of the primer extension analysis of 16S rRNA bases modified with DMS as a function of the indicated time (ms) elapsed after mixing free 30S subunits or 30S-IF3 complexes with 50S subunits. The other lanes indicated are the following: 16S rRNA sequencing (A and G), 16S rRNA without DMS (K), and 30S or 30S-IF3 modified in the absence of 50S (C). The graphs on the right side display the densitometric quantification of the band intensities of bases modified in 30S (black tracings) and in 30S-IF3 (colored tracings). In all panels, the reactivity of the individual bases as a function of time elapsed after mixing 30S or 30S-IF3 with 50S is expressed as the percent of the reactivity displayed before mixing. The positions of these bases are indicated with the same colors in the three-dimensional structures of 16S rRNA within the 30S subunit. The modified bases are the following: (A) A790, (B) A704, (C) C750, (D) A702, and (E) A759. All controls and experiments were repeated at least three times, and the standard deviation between the normalized intensity of bands was used to generate error bars. Molecular Cell 2007 25, 285-296DOI: (10.1016/j.molcel.2006.12.011) Copyright © 2007 Elsevier Inc. Terms and Conditions