Functional and Structural Studies on Artemia Polypeptide Chain Initiation Factor 2. Cloning and Sequencing of eIF-2α cDNA

Abstract

The eukaryotic polypeptide chain initiation factor 2 (eIF-2) plays a significant role in the regulation of protein synthesis [1,2]. The first step in polypeptide chain initiation is the formation of a ternary complex containing eIF-2, GTP and the initiator tRNA. This complex is transferred to a 40S ribosomal subunit [1–4] which later combines with a 60S ribosomal subunit and other initiation factors to form an 80S initiation complex, thereby setting the stage for polypeptide chain elongation. Upon formation of the 80S initiation complex, GTP is hydrolyzed and eIF-2 is released as the eIF-2·GDP binary complex [5,6]. In mammalian systems, this binary complex is stable in the presence of Mg2+ and is functionally inactive [7,8]. Regeneration of eIF-2·GTP·Met-tRNAf species require the guanine nucleotide exchange factor (GEF), which facilitates the exchange of elF-2-bound GDP for GTP and facilitates the recycling of eIF-2 [9–12]. It is at this point in the eIF-2 cycle (Fig. 1) that regulation of polypeptide chain initiation occurs. Most of our understanding about this step in protein synthesis in eukaryotes is derived from studies with rabbit reticulocyte lysates. We have previously observed that dormant and developing embryos of Artemia contain equivalent amounts of eIF-2 activity [13]. Similar to reticulocyte eIF-2, the Artemia factor eIF-2 has 100 to 300-fold higher affinity for GDP than GTP [13] and its α-subunit gets phosphorylated by the rabbit reticulocyte heme-controlled repressor (HCR) [14].