The roles of initiation factor 2 and guanosine triphosphate in initiation of protein synthesis

Abstract

The role of IF2 from Escherichia coli was studied in vitro using a system for protein synthesis with purified components. Stopped flow experiments with light scattering show that IF2 in complex with guanosine triphosphate (GTP) or a non-cleavable GTP analogue (GDPNP), but not with guanosine diphosphate (GDP), promotes fast association of ribosomal subunits during initiation. Biochemical experiments show that IF2 promotes fast formation of the first peptide bond in the presence of GTP, but not GDPNP or GDP, and that IF2-GDPNP binds strongly to post-initiation ribosomes. We conclude that the GTP form of IF2 accelerates formation of the 70S ribosome from subunits and that GTP hydrolysis accelerates release of IF2 from the 70S ribosome. The results of a recent report, suggesting that GTP and GDP promote initiation equally fast, have been addressed. Our data, indicating that eIF5B and IF2 have similar functions, are used to rationalize the phenotypes of GTPase-deficient mutants of eIF5B and IF2.

Fig. 1. The effects of fMet-tRNA^fMet and 30S subunits on the affinity of GDP or GTP to IF2. The extent of GDP (A) or GTP (B) binding to IF2 was measured by nitrocellulose filtration as a function of G-nucleotide concentration in the presence of combinations of fMet-tRNA^fMet and 30S subunits containing mMFTI mRNA and IF1. Open circles: with neither fMet-tRNA^fMet nor 30S; closed circles: with 30S; open squares: with fMet-tRNA^fMet; closed squares: with fMet-tRNA^fMet and 30S.

Fig. 2. The effects of G-nucleotides on the rate of association of 30S pre-initiation complexes with 50S subunits. The extent of 70S complex formation was monitored as a function of time by light scattering after rapid mixing of pre-initiation 30S complexes with 50S subunits in a stopped-flow instrument. Time curves were obtained with GTP (A), GDPNP (B) or GDP (C).

Fig. 3. The effects of G-nucleotides and IF2 concentration on the rate of peptidyl transfer, starting from pre-initiation 30S complexes and 50S subunits. Subunits were mixed in the presence of IFC and either GTP (diamonds), GDP (triangles) or GDPNP (squares). The reaction mixture contained either 2.5 pmol IF2 (A) or 120 pmol IF2 (B) per 50 pmol of ribosomes. The extent of ribosome initiation was monitored by dipeptide formation. Ternary complexes (EF-Tu–GTP–Phe-tRNAP^he) were added at different time points after subunit mixing, and the peptidyl-transfer reaction was quenched with 50% formic acid after 5 s.

Fig. 4. Peptide bond formation in the presence of IF2 and GDPNP. The 70S complexes were assembled by mixing 0.4 µM 50S subunits and 0.4 µM 30S pre-initiation complexes in the presence of 0.04 µM IF2 and 10 µM GTP. After 10 min of incubation at 37C, either 0.5 µM IF2 and 0.2 mM GDPNP (closed squares, ‘low IF2-GDPNP’ mix), or 1.5 µM IF2 and 0.2 mM GDPNP (open squares, ‘high IF2-GDPNP’ mix), or an equivalent volume of polymix buffer (open diamonds) were added to these 70S complexes. The incubation then continued for 1 min more, after which EF-Tu–GTP–Phe-tRNA ternary complexes (A) or puromycin (B) were added to all mixes and the [³H]fMet-Phe or [³H]fMet-Pur formation was followed as a function of time. Ternary complexes (A) were also added to the ‘high’ (open circles) and ‘low’ (closed circles) IF2-GDPNP mixes, together with a large amount of GTP (1.4 mM final concentration). Puromycin (B) was also added to the ‘low IF2-GDPNP’ mix together with a large amount of GTP (closed circles) or GDP (closed triangles). As a control, 70S complexes were also assembled from 0.4 µM 50S subunits and 0.4 µM 30S pre- initiation complexes in the presence of 0.5 µM IF2 and 0.2 mM GTP. After incubation for 11 min, the extent of complex formation was checked by the addition of ternary complexes (A, closed diamonds). Note that open diamonds in (B) come from two independent experiments.

Fig. 5. IF2 dissociation from 70S initiation complex. The 70S initiation complexes were assembled by mixing 0.4 µM 50S subunits and 0.4 µM 30S pre-initiation complexes in the presence of 0.02 µM His-tagged cold IF2 and 10 µM GTP. After 15 min of incubation at 37°C, GDPNP was added to 0.5 mM final concentration. Then [³⁵S]IF2 (His tagged) was added to the 0.4 µM final concentration. After 2 min of incubation, an excess of cold His-tagged IF2 (1.2 µM final concentration) was added to the reaction mix to start the exchange between hot and cold IF2 (closed squares). The exchange was also studied in the presence of 1 µM of ternary complexes (closed diamonds). Aliquots of the reaction were taken at the indicated times and the amount of ribosome-bound hot IF2 was determined as described in Materials and methods. The slope of the plot of the logarithm of this amount versus time in (A) gives the rate of IF2 dissociation. The extend of dipeptide formation in the reaction mix containing ternary complex was also measured. The slope of Log[Dip(max)-Dip(t)] (closed diamonds) versus time gives the rate of dipeptide formation in the reaction mix (B).

Fig. 6. The effects of temperature and [Mg²⁺] on the rate of peptidyl transfer, starting from pre-initiation 30S complexes and 50S subunits, and on the exchange of GTP for GDP on IF2 in the pre-initiation 30S complex. (A) The extent of ribosome initiation was monitored as in Figure 3 in the presence of IF2 and GTP (closed diamonds), IF2 and GDP (closed triangles), IF2 and GDPNP (closed squares), or in the absence of IF2 (open circles). The reaction mixture at 20°C contained 120 pmol IF2 per 50 pmol ribosome and 7 mM Mg(OAc)₂. (B) The extent of ribosome initiation was monitored as in Figure 3 in the presence of IF2 and GTP (closed diamonds), IF2 and GDP (closed triangles), and IF2 and GDP but with the 30S pre-initiation complex first pre-incubated (10 min) with 70 µM of GTP and then (1.5 min) with 2 mM GDP (closed circles). (C) IF2, alone or with 30S in complex with mRNA, fMet-tRNA^fMet, IF1 and IF3, was pre-incubated (10 min) with [³H]GTP (10 µM), and the amount of [³H]GTP remaining on IF2 after 1 min incubation following the addition of varying concentrations of unlabelled GDP was monitored by nitrocellulose filtration: closed diamonds, 5 mM Mg²⁺ at 37°C; closed squares, 7 mM Mg²⁺ at 20°C; closed triangles, 5 mM Mg²⁺ at 37°C with 30S complex; closed circles 7 mM Mg²⁺ at 20°C with 30S complex.