How Initiation Factors Maximize the Accuracy of tRNA Selection in Initiation of Bacterial Protein Synthesis  Ayman Antoun, Michael Y. Pavlov, Martin Lovmar, Måns Ehrenberg  Molecular Cell  Volume 23, Issue 2, Pages 183-193 (July 2006) DOI: 10.1016/j.molcel.2006.05.030 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Effects of Initiation Factors on the Kinetics of fMet-tRNAfMet, Met-tRNAfMet, and Phe-tRNAPhe Interaction with the 30S Ribosomal Subunit (A) Association of Met-tRNAfMet or Phe-tRNAPhe to the 30S·mRNA·IF1·IF2 complex, measured by NC filtration. (Inset) Association of fMet-tRNAfMet to the same 30S complex, measured by light scattering. (B) Association of Phe-tRNAPhe to mRNA-programmed 30S subunits in the presence of IF1 and IF2, of only IF1, of only IF2, or of no factors, measured by NC filtration. The association of fMet-tRNAfMet in the absence of initiation factors is shown for comparison. (C) Dissociation of [3H]Met-tRNAfMet from preassembled 30S complexes containing IF3 and different combinations of IF1 and IF2. Dissociation was measured in a chase experiment by NC filtration. (D) Dissociation of [3H]fMet-tRNAfMet (filled symbols) or [3H]Phe-tRNAPhe (open symbols) from 30S subunits programmed with xMFTI mRNA or xFMTI mRNA in the absence of initiation factors. Dissociation was measured in a chase experiment by NC filtration. Molecular Cell 2006 23, 183-193DOI: (10.1016/j.molcel.2006.05.030) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Docking of the 50S Subunit to 30S Preinitiation Complexes Assembled with Different tRNAs and Combinations of Initiation Factors Measured by Light Scattering (A) 30S preinitiation complexes contained no initiation factors. (B) 30S preinitiation complexes contained IF1 and IF2 (closed symbols) or only IF2 (open symbols). (Inset) The same curves in a short time range. (C) 30S preinitiation complexes contained IF1 and IF3 (closed symbols) or IF3 (open symbols). (D) 30S preinitiation complexes contained IF1, IF2, and IF3 (closed symbols) or IF2 and IF3 (open symbols). (Inset) The same curves in a short time range. Molecular Cell 2006 23, 183-193DOI: (10.1016/j.molcel.2006.05.030) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Cartoon of Initiation of Protein Synthesis with Definitions of Elemental Rate Constants (A) Different tRNAs associate to mRNA-programmed 30S subunits equipped with three initiation factors with the association rate constant ka,t and dissociate with the dissociation rate constant kd,t. IF3 dissociates from the 30S preinitiation complex with the rate kd,3, after which the 50S subunit can dock to an IF3-free 30S preinitiation complex with the association rate constant ka,r. Alternatively, IF3 can rebind to the 30S preinitiation complex with the association rate constant ka,3. Rate constants ka,t, kd,t, kd,3, and ka,r are different for fMet-tRNAfMet, Met-tRNAfMet, and Phe-tRNAPhe. (B) The same as (A) but in the absence of IF3. In this case, the rate of tRNA dissociation (kd,t) is much smaller than the rate ka,r·[50S] of 50S docking. Thus, any tRNA bound to the 30S preinitiation complex has very little chance to dissociate and therefore always ends up in the 70S initiation complex. (C) Cartoon illustrating how a varying distance between the SD sequence and an initiation codon affects the kinetics of tRNA interaction with the 30S subunit. The 30S subunit has four states: 30S0 or T:30S0, where the initiator codon is off the P site with or without tRNA, respectively, and 30Sm or T:30Sm, where the initiator codon is in the P site with or without tRNA, respectively. 30S0 and T:30S0 are converted to 30Sm and T:30Sm with rate constants ks and kts, respectively. 30Sm and T:30Sm are converted to 30S0 with rate constants ks′ and kts′, respectively. tRNA associates with 30S0 and 30Sm with the same rate constant ka and dissociates from T:30S0 and T:30Sm with rate constants kd′ and kd, respectively. When T:30S0 and T:30Sm are in rapid equilibrium (ks/ks′≪1andkts/kts′≫1), then the measured association rate constant kon = ka and the measured dissociation rate constant koff=kd′(kts′/kts). Thus, optimizing the SD-initiator codon distance under these conditions maximizes kts/kts′, minimizes koff, and leaves kon unaltered. Molecular Cell 2006 23, 183-193DOI: (10.1016/j.molcel.2006.05.030) Copyright © 2006 Elsevier Inc. Terms and Conditions