Polypeptide chain initiation in eukaryotes: reversibility of the ternary complex-forming reaction.

Abstract

In the last step of polypeptide chain initiation in eukaryotes, the interaction of the 40S preinitiation complex eIF-2 GTP Met-tRNAj 4OS [the complex between the 40S ribosomal subunit and the ternary complex containing equimolar amounts of eukaryotic initiation factor 2 (eIF-2), GTP, and eukaryotic initiator methionyl tRNA (Met-tRNA;)] with a 60S ribosomal subunit in the presence of mRNA, cap binding protein (with "capped" messengers), ATP, and the initiation factors eIF-3, eIF-4a, -4b, -4c, and eIF-5, results in the formation of an 80S initiation complex (MettRNAj'80S'mRNA) with concomitant hydrolysis of GTP and liberation of eIF-2 for recycling in subsequent initiation events. However, at physiological Mg2+ concentrations, GDP is known to have '100-fold greater affinity than GTP for eIF-2 and eIF-2 is believed to be released in the form of an eIF-2'GDP complex. Previously, we have shown that initiation factor SP (for eIF-2-stimulating protein) promotes the exchange of eIF-2-bound GDP for GTP and catalyzes ternary complex formation in the presence of Met-tRNA;. Binding of GDP by eIF-2 is indeed so tight that, as we now show, homogeneous preparations of eIF-2 contain upward of 0.5 mol of GDP/mol of eIF-2. We further show that, in the presence of Mg2+ and catalytic amounts of SP, ternary complex formation conforms to the overall reversible reaction eIF-2'GDP + GTP + Met-tRNA, = eIF-2 GTP'Met-tRNA; + GDP.The first step of polypeptide chain initiation in eukaryotes is the formation of a ternary complex containing equimolar amounts of eukaryotic initiation factor 2 (eIF-2), GTP, and eukaryotic initiator methionyl tRNA (Met-tRNAi). This complex binds to a 40S ribosomal subunit, giving rise to a 40S preinitiation complex. In the presence of a 60S ribosomal subunit, mRNA, additional factors, and ATP, an 80S initiation complex is formed, setting the stage for chain elongation. Formation of the 80S complex is accompanied by GTP hydrolysis and eIF-2 release (see the diagram in ref. 1). Ternary complex formation is inhibited by Mg2+ (2), which, however, is required for the ensuing steps (3). In the presence of Mg2+, eIF-2 has at least 100-fold higher affinity for GDP than for GTP (4), suggesting that the factor is released as an eIF-2-GDP complex on completion of each initiation round. In the presence of Mg2+, formation of a ternary complex (which contains GTP) requires, therefore, a mechanism for exchange of eIF-2-bound GDP for GTP (5, 6). This exchange is catalyzed by a factor termed SP [for eIF-2-stimulating protein (6)] or GEF [for GDP exchange (7)]. The strong similarity of ternary complex formation in eukaryotic chain initiation and prokaryotic chain elongation has been pointed out (6). It is now apparent that eIF-2 and SP are the counterparts of the chain elongation factors EF-Tu and EF-Ts, respectively (6). GDP (unpublished data), providing direct evidence for reversal of reaction a. In this paper, we show that, unlike eIF-2'SP, eIF-2 is isolated in the form of an eIF-2-GDP complex and present evidence for...

Protein synthesis in Drosophila melanogaster embryos

“…It is remarkable that the phosphorylation sites in the smallest subunits of Drosophila and rabbit reticulocyte [37] eIF-2 are localized in a terminal fragment which suggests they are closely related. On the other hand, we have shown that, again as in the case of rabbit reticulocytes eIF-2 [38], the Drosophila factor can be isolated as an eIF-2 . GDP complex which is compatible with the notion that the factor has a high affinity for GDP.…”

“…Samples contained in a final volume of 25 pl, 20 mM Hepes buffer pH 7.6, 100 mM KCl, 10 pM Mg(CH3C00)2, 1 mM dithiothreitol, 25 pg bovine serum albumin, 1.8 pM [3H]GDP (8900 cpm/pmol, Amersham) and eIF-2 (which, like reticulocyte eIF-2 [38], contains bound GDP) in the amounts indicated in each experiment. After incubation for 6 min at 25"C, samples were processed as described for ternary complex assay.…”

Identification of Caspase 3-mediated Cleavage and Functional Alteration of Eukaryotic Initiation Factor 2α in Apoptosis

“…In light of this result, the modest inhibition of eIF2␣ cleavage observed upon eIF2␣ phosphorylation in Fig. 7A is most likely due to the fact that GDP remains bound to a small fraction of purified eIF2 (26). As such, the Mg 2ϩ added to the kinase reaction would stabilize the binding of this GDP to eIF2 and inhibit its cleavage by caspases.…”

Inhibition of protein synthesis by the β‐subunit of spectrin

“…In the same figure the inhibitory effect of the reticulocyte heme-controlled eIF-2cu kinase is shown for comparison. This kinase phosphorylates the cy-or smallest subunit of peptide initiation factor 2, eIF&, and thereby inhibits binding of MettRNAf to 40 S ribosomal subunits [15] apparently by preventing the exchange of GTP for GDP from the GDP -eIF-2 complex [16]. On the basis of the weight of protein added to the lysate reaction mixture &spectrin is about half as inhibitory as the kinase, as judged from the initial slope of the inhibition curves.…”