GTP interacts with the γ‐subunit of eukaryotic initiation factor eIF‐2

Abstract

Eukaryotic initiation factor eIF-2 is an oligorneric protein consisting of three different subunits. During initiation of protein synthesis eIF-2 interacts with GTP, Met-tRNAland 40 S ribosomal subunit. By afftnity labeling with a photo-reactive GTP analogue it was shown that in the binary complex [eIF-2. GTP] GTP is in contact with the y-subunit of eIF-2.

Shape and location of eukaryotic initiation factor eIF‐2 on the 40s ribosomal subunit of rat liver

“…subunit, are as active as complete eIF-2 in Met-tRNA, binding to 40s ribosomal subunits [38-421. (c) Because of its basic nature [18] the y subunit is the main candidate to contribute to the binding strength of the factor to the 40s subunit, and it seems to be logical that GTP was found to interact with the y subunit [16], if one considers that GTP hydrolysis is necessary for the release of eIF-2 from the 40s subunit (reviewed in [4,51).…”

“…Initiation factor eIF-2 was prepared from rat liver according to Nygard et al [15] with slight modifications briefly described by Kurzchalia et al [16]. The whole procedure was performed at about 2°C and in the presence of 0.2 mM phenylmethylsulfonyl fluoride to prevent proteolytic degradation.…”

“…The gradients were fractionated, and 40-pl aliquots were counted for radioactivity. Fractions indicated by arrows were taken as dimeric immune complcxes and investigated in the electron microscope (10) 28 (7) 378 (100) anti-(eIF-27) 2 (1) 4 (2) 79 (30) 57 (22) 105 (40) 18 (7) 265 (100) anti-(eIF-2) 25 (16) 35 (22) 19 (12) 21 (13) 54 (35) 2 (1) 156 (100) Fig. 5.…”

GTP-dependent Recognition of the Methionine Moiety on Initiator tRNA by Translation Factor eIF2

“…8 The gamma subunit of eIF2 is thought to bind both the initiator tRNA and the guanine nucleotide. 21,36,37 The structures of the wild-type protein in the absence of guanine nucleotide and the presence of GDP were determined, as were structures of a mutant form of the protein, G235D (thought to be in or near the MettRNA i binding site), in the absence of ligand and the presence of both GDP and GDPNP. The archaeal gamma subunit bears striking sequence and structural similarities to elongation factor EFTu/eEF1A.…”

Initiation of protein synthesis in mammalian cells

“…Surprisingly, there are still significant gaps in our knowledge ofeven the most basic mechanisms involved in the formation of the ternary complex. It was originally suggested that the GTP ligand binds to the ac subunit and Met-tRNAf to the , subunit [19], but more recent work using affinity labelling with photo-reactive analogues of GTP has indicated that guanine nucleotides bind either to the y [20] or the , (W. C. Merrick, personal communication) subunits. In the latter study the analytical system used was capable of distinguishing unambiguously between the and y subunits, and both studies included controls demonstrating specificity of association.…”