The nucleotide-binding site of bacterial translation initiation factor 2 (IF2) as a metabolic sensor

Abstract

Translational initiation factor 2 (IF2) is a guanine nucleotide-binding protein that can bind guanosine 3',5'-(bis) diphosphate (ppGpp), an alarmone involved in stringent response in bacteria. In cells growing under optimal conditions, the GTP concentration is very high, and that of ppGpp very low. However, under stress conditions, the GTP concentration may decline by as much as 50%, and that of ppGpp can attain levels comparable to those of GTP. Here we show that IF2 binds ppGpp at the same nucleotide-binding site and with similar affinity as GTP. Thus, GTP and the alarmone ppGpp can be considered two alternative physiologically relevant IF2 ligands. ppGpp interferes with IF2-dependent initiation complex formation, severely inhibits initiation dipeptide formation, and blocks the initiation step of translation. Our data suggest that IF2 has the properties of a cellular metabolic sensor and regulator that oscillates between an active GTP-bound form under conditions allowing active protein syntheses and an inactive ppGpp-bound form when shortage of nutrients would be detrimental, if not accompanied by slackening of this synthesis.

Fig. 1.

IF2 binding of GTP and ppGpp. (A) Concentration dependence of k_app of mant-GTP–IF2 interaction. (B) Time courses of mant-GTP dissociation (5 μM) from IF2 upon addition of 25 μM GTP (red), 25 μM ppGpp (blue), or buffer (green).

Fig. 2.

Interaction of IF2G2 with GDP and ppGpp monitored by NMR spectroscopy. (A) Overlay of the ¹⁵N-HSQC spectra of IF2G2·GDP (black) and IF2G2·ppGpp (red). The assigned residues close to guanosine nucleotide are indicated. (Inset) Downfield region of the 1D ¹H NMR watergate spectra of the same complexes. (B) Expansions of the 2D spectral regions displaying the most pronounced differences between IF2G2·GDP and IF2G2·ppGpp. (C–E) Views of the NMR 3D structure of IF2G2·ppGpp (7). The protein is represented by ribbons and ppGpp by “balls and sticks,” with the 3′ diphosphate indicated by red arrows.

Fig. 3.

Effect of ppGpp and GDP on IF2-dependent 30S initiation complex formation. (A) fMet-tRNA-binding kinetics to mRNA-programmed 30S subunits measured by rapid filtration (see Materials and Methods) in the presence of 1 mM GTP (▴), 0.25 mM GTP + 0.75 mM GDP (●), and 0.25 mM GTP + 0.75 mM ppGpp (■). (B) Level of fMet-tRNA bound as a function of ligand concentration. Different ligand concentrations in mixtures containing constant concentrations of guanosine nucleotides were obtained, keeping amounts of all 30S initiation complex components (30S, mRNA, fMet-tRNA, IF1, and IF2) constant but varying the reaction volumes (from 15 to 300 μl). The actual 30S concentrations ranged from 25 to 500 nM, and the stoichiometric ratios ligand/30S were IF1, IF2, and IF3 = 1.5; fMet-tRNA and mRNA = 2.0. The reciprocal of 30S subunits concentration in the reaction mixtures is indicated in the abscissa. The levels of 30S-bound fMet-tRNA 30S subunit in the presence of GDP (0.5 mM) + GTP (0.25 mM) (●) and ppGpp (0.5 mM) + GTP (0.25 mM) (■) are expressed as % inhibition with respect to the controls containing GTP (0.75 mM) alone.

Fig. 4.

Effect of GDP and ppGpp on the kinetics of IF2-dependent initiation dipeptide formation. IF2-dependent formation of initiation dipeptide (fMet-Phe) in the presence of 1 mM GTP (▴); GTP + GDP, 0.5 mM each (●); and GTP + ppGpp (■), 0.5 mM each (A), and a constant concentration (0.25 mM) of GTP and increasing concentrations of ppGpp (0 mM, ▴; 0.10 mM, ●; 0.25 mM, ♦; 0.50 mM, ■; 0.75 mM, ▾; and 1.0 mM, ∗) (B). The amount of initiation dipeptide formed is expressed as fMet-Phe formed/fMet-tRNA bound in the 30SIC. (C) Variation of the k_app of dipeptide formation as a function of [ppGpp]. (D) Dixon plot showing the linear dependence of 1/k_app as a function of increasing [ppGpp]. The slope is equal to K_m/(V_max·[S]·k_i). Initiation dipeptide formation was analyzed by quench-flow experiments at 37°C, as described in Materials and Methods.

Fig. 5.

Inhibition of mRNA translation by various guanosine nucleotides. Translation in cell-free systems containing a constant concentration of GTP (0.25 mM) and programmed with 027AUGmRNA (closed symbols) and 027AUUmRNA (open symbols) is plotted as a function of the indicated concentrations of ppGpp (A), GDP (B), and GDPNP (C). The levels of translation indicated on the ordinate are normalized for the level of translation (taken as 1) obtained in the absence of added guanine nucleotide inhibitors. (D) Translation inhibition ratios (027AUUmRNA/027AUGmRNA) plotted as a function of the indicated concentrations of ppGpp (■), GDP (●), GDPNP (▾), and GTP (▴).

Fig. 6.

Schematic representation of the postulated regulatory circuits involving IF2 and ppGpp. (Upper) The essential steps of translation initiation: formation of 30SIC in the presence of IF2-GTP (▴), association of 50S subunit and formation of 70SIC, GTP (▴) hydrolysis to GDP (), IF2 dissociation (), and initiation dipeptide formation. Amino acid starvation during elongation triggers RelA-dependent synthesis of ppGpp (■), which inhibits stable RNA transcription (Lower) and, as shown in this study, the initiation functions of IF2 (30SIC formation and initiation dipeptide formation). A possible function of translation initiation intermediates containing IF2-ppGpp () in feedback inhibition of stable RNA transcription is suggested by earlier reports that components of the translation initiation apparatus play an active part in this regulation. In particular, IF2, fMet-tRNA, and ppGpp were found to interact with the RNA polymerase and influence its activity at stable RNA promoters (38, 39), whereas IF2 (40), IF3 (41), and initiation-competent 30S subunits (42) were shown to be required for feedback repression of stable RNA transcription.