Mechanism of polypeptide chain initiation in eukaryotes and its control by phosphorylation of the alpha subunit of initiation factor 2.

Abstract

Earlier, eukaryotic initiation factor 2(eIF-2)-stimulating protein (SP) was isolated as a homogeneous complex with eIF-2 (eIF-2.sbd.SP) and, in the presence of Mg2+, eIF-2.sbd.SP promoted formation of a ternary complex with GTP and eukaryotic initiator methionyl tRNA (Met-tRNAi) (eIF-2-GTP-Met-tRNAi) catalytically. SP-bound eIF-2 exchanged with eIF-2 (eIF-2 exchange). Furthermore, in the presence of Mg2+, eIF-2.sbd.SP catalyzes the exchange of eIF-2-bound [3H]GDP with unlabeled GDP or GTP (GDP exchange) and the release of [3H]GDP when the ternary complex is formed from eIF-2.sbd.[3H]GDP, GTP, and [35S]Met-tRNAi. All these reactions are blocked by .alpha.-subunit, but not by .beta.-subunit, phosphorylation of eIF-2. The eIF-2 and GDP exchanges are compatible with the reaction eIF-2.sbd.GDP + SP .dblarw. EIF-2.sbd.SP + GDP reminiscent of the exchange between the Tu and Ts components of prokaryotic elongation factor 1 (EF-Tu and EF-Ts, respectively) EF-Tu.sbd.GDP + EF-Ts .dblarw. EF-Tu.sbd.EF-Ts + GDP. Due to the high affinity of GDP (.simeq. 100 times greater than that of GTP) for eIF-2, 40S (eIF-2.sbd.GTP.sbd.Met-tRNAi.sbd.40S) to 80S (Met-tRNAi.sbd.mRNA.sbd.80S) initiation complex conversion, which is accompanied by GTP hydrolysis, probably releases eIF-2 as eIF-2.sbd.GDP. In the presence of Mg2+, GDP binding probably restricts the availability of eIF-2 for chain initiation and SP relieves this restriction in a catalytic fashion, provided that the .alpha. subunit of eIF-2 is not phosphorylated.