The ribosome-bound initiation factor 2 recruits initiator tRNA to the 30S initiation complex

Abstract

Bacterial translation initiation factor 2 (IF2) is a GTPase that promotes the binding of the initiator fMet-tRNA(fMet) to the 30S ribosomal subunit. It is often assumed that IF2 delivers fMet-tRNA(fMet) to the ribosome in a ternary complex, IF2.GTP.fMet-tRNA(fMet). By using rapid kinetic techniques, we show here that binding of IF2.GTP to the 30S ribosomal subunit precedes and is independent of fMet-tRNA(fMet) binding. The ternary complex formed in solution by IF2.GTP and fMet-tRNA is unstable and dissociates before IF2.GTP and, subsequently, fMet-tRNA(fMet) bind to the 30S subunit. Ribosome-bound IF2 might accelerate the recruitment of fMet-tRNA(fMet) to the 30S initiation complex by providing anchoring interactions or inducing a favourable ribosome conformation. The mechanism of action of IF2 seems to be different from that of tRNA carriers such as EF-Tu, SelB and eukaryotic initiation factor 2 (eIF2), instead resembling that of eIF5B, the eukaryotic subunit association factor.

Figure 1

A scheme of the interactions between IF2·GTP, fMet-tRNA^fMet and the complex 30S·mRNA·IF1·IF3 (denoted as 30S^*). GTP bound to IF2 is omitted for simplicity. fMet-tRNA^fMet can bind to the 30S^* complex in the ternary complex with IF2·GTP (steps 1/3 pathway) or to the IF2·GTP·30S^* complex (steps 2/4 pathway). IF, initiation factor.

Figure 2

The kinetics of IF2·GTP·fMet-tRNA^fMet complex formation. (A) Binding of IF2(Atto) (0.2 μM) with GTP (0.25 mM) to fMet-tRNA^fMet(QSY) (2 μM; trace-1) or to unlabelled fMet-tRNA^fMet (2 μM; trace-2). (B) Concentration dependence of k_app. Values of k_app were estimated by one-exponential fitting of the time courses obtained at increasing concentrations of fMet-tRNA^fMet(QSY). Linear regression yielded k₁=48±8 μM⁻¹ s⁻¹ and k₋₁=43±10 s⁻¹, assuming a one-step binding model. (C) Dissociation of the IF2·GTP·fMet-tRNA^fMet complex. IF2(Atto) (0.2 μM) and fMet-tRNA^fMet(QSY) (1 μM) were pre-incubated and mixed with unlabelled IF2 (2 μM; trace-1) or buffer A (trace-2) in the presence of GTP (0.25 mM). Exponential fitting of trace-1 gives k₋₁=40±5 s⁻¹. Numerical integration (continuous lines) yielded k₁=40±3 μM⁻¹ s⁻¹ and k₋₁=38±2 s⁻¹, assuming a one-step binding model. (D) The kinetic parameters of ternary complex formation. IF, initiation factor.

Figure 3

IF2 binding to the 30S^* complex. (A) The time course of IF2(Atto) (0.025 μM) binding to the 30S subunit (0.075 μM) in the presence of mRNA (0.75 μM), IF1 (0.75 μM), IF3(Alx) (0.75 μM) and GTP (0.25 mM; trace-1). Controls were performed with unlabelled IF2 (acceptor only, trace-2) or unlabelled IF3 (donor only, trace-3). Binding was monitored by FRET from IF2(Atto) to IF3(Alx). k_app21=30±3 s⁻¹ and k_app22=3±0.3 s⁻¹ were estimated by two-exponential fitting. (B) The concentration dependence of k_app21. IF2(Atto) (0.025 μM) was mixed with the 30S^* complex at increasing concentrations. The rate constants k₂₁=200±20 μM⁻¹ s⁻¹ and k₋₂₁=15±3 s⁻¹ were obtained from the linear fit. (C) Dissociation of IF2(Atto) (0.025 μM) from the 30S·mRNA·IF1·IF3(Alx) complex (0.25 μM) on mixing with unlabelled IF2 (2 μM; trace-1) or buffer A (trace-2). Dissociation rate constant k₋₂₂=1±0.1 s⁻¹. (D) The two-step binding mechanism. FRET, fluorescence resonance energy transfer; IF, initiation factor.

Figure 4

The kinetics of fMet-tRNA^fMet binding to the 30S subunit. (A) The time courses of fMet-tRNA^fMet binding to the 30S^* complex. IF2 (0.15 μM, concentrations after mixing) was preincubated with fMet-tRNA^fMet(Flu) (0.5 μM) and GTP (0.5 mM), and mixed with 30S subunits (0.075 μM), mRNA (0.3 μM), IF1 (0.15 μM), IF3(Alx) (0.15 μM) and GTP (0.5 μM; trace-1). Alternatively, fMet-tRNA^fMet(Flu) was mixed with 30S^*·IF2·GTP (trace-2). Controls were performed with unlabelled fMet-tRNA^fMet (acceptor only, trace-3) or unlabelled IF3 (donor only, trace-4). (B) The concentration dependence of fMet-tRNA^fMet binding to the 30S^* complex. Closed symbols, IF2 preincubated with fMet-tRNA^fMet; open symbols, IF2 preincubated with the 30S^* complex; circles, rapid filtration; triangles, stopped-flow. (C) Dissociation of fMet-tRNA^fMet from the 30S IC. The complex was formed by incubation of 30S subunits (0.05 μM) with 002mRNA (0.2 μM), IF1 (0.1 μM), IF2 (0.1 μM), IF3(Alx) (0.1 μM), f[³H]Met-tRNA^fMet(Flu) (0.15 μM) and GTP (0.25 mM). FRET was monitored from f[³H]Met-tRNA^fMet(Flu) to IF3(Alx). Exchange of f[³H]Met-tRNA^fMet(Flu) was induced by the addition of unlabelled fMet-tRNA^fMet (2.25 μM) alone (trace-1) or together with IF2 (2 μM; trace-2). In the control experiment, buffer A was added (trace-3). Closed circles, 30S-bound f[³H]Met-tRNA^fMet determined by nitrocellulose filtration. Single-exponential fitting (trace-1/2) yielded a dissociation rate constant of 0.004±0.001 s⁻¹. (D) The concentration dependence of binding to the 30S^* complex of IF2·GTP (0.05 μM) preincubated with fMet-tRNA^fMet (1.5 μM). FRET was monitored from IF2(Atto) to IF3(Alx). In addition, a fast binding step was observed due to binding of free IF2 to the 30^* complex (data not shown). FRET, fluorescence resonance energy transfer; IC, initiation complex; IF, initiation factor.

Figure 5

The pathway of IF2·GTP and fMet-tRNA^fMet binding to the 30S^* complex. The main pathway is shown vertically. The formation of the ternary complex IF2·GTP·fMet-tRNA^fMet, which is probably an insignificant side reaction under normal conditions, might take place under conditions of 30S subunit shortage, perhaps acting as a storage complex. IC, initiation complex; IF, initiation factor.