The A1 · U72 Base Pair Conserved in Eukaryotic Initiator tRNAs Is Important Specifically for Binding to the Eukaryotic Translation Initiation Factor eIF2

Abstract

The formation of a specific ternary complex between eukaryotic initiation factor 2 (eIF2), the initiator methionyl-tRNA (Met-tRNA), and GTP is a critical step in translation initiation in the cytoplasmic proteinsynthesizing system of eukaryotes. We show that the A1 ⅐ U72 base pair conserved at the end of the acceptor stem in eukaryotic and archaebacterial initiator methionine tRNAs plays an important role in this interaction. We changed the A1 ⅐ U72 base pair of the human initiator tRNA to G1 ⅐ C72 and expressed the wild-type and mutant tRNA genes in the yeast Saccharomyces cerevisiae by using constructs previously developed in our laboratory for expression of the human initiator tRNA gene in yeasts. We show that both the wild-type and mutant human initiator tRNAs are aminoacylated well in vivo. We have isolated the wild-type and mutant human initiator tRNAs in substantially pure form, free of the yeast initiator tRNA, and have analyzed their properties in vitro. The G1 ⅐ C72 mutation affects specifically the binding affinity of eIF2 for the initiator tRNA. It has no effect on the subsequent formation of 40S or 80S ribosome initiator Met-tRNA-AUG initiation complexes in vitro or on the puromycin reactivity of the Met-tRNA in the 80S initiation complex.A special methionine tRNA is used to initiate protein synthesis in all organisms studied. Of the two classes of methionyltRNA (Met-tRNA), the initiator is used for the initiation of protein synthesis whereas the elongator is used for insertion of methionine into internal peptide bonds (27,36). Eubacteria, mitochondria, and chloroplasts initiate protein synthesis by using formyl-Met-tRNA, whereas the cytoplasmic system of eukaryotes uses 45).Because of their unique function, initiator tRNAs possess several properties which are distinct from those of elongator tRNAs. For eukaryotic initiator tRNAs, these include (i) the highly specific formation of a ternary complex between the initiator Met-tRNA, eukaryotic initiation factor 2 (eIF2), and GTP; (ii) the binding of the initiator Met-tRNA to the P site on the 80S ribosome; and (iii) exclusion of the initiator MettRNA from the ribosomal A site. In contrast, the elongator tRNAs form a ternary complex with the elongation factor EF-1 and GTP and then bind to the ribosomal A site.The initiator tRNA also plays an important role in the formation of the 40S and 80S ribosomal initiation complexes (21,33,46). Once the eIF2 ⅐ GTP ⅐ Met-tRNA ternary complex is formed, it binds to the 40S ribosomal subunit. This new complex, consisting of the 40S ribosome, eIF2, GTP, and MettRNA, then binds to the capped 5Ј end of mRNA in a reaction requiring several initiation factors. The 40S ribosome containing the bound eIF2 ⅐ GTP ⅐ Met-tRNA ternary complex then scans the mRNA until it reaches the AUG codon (28). Donahue and coworkers (8, 12) have shown that in Saccharomyces cerevisiae, the initiator Met-tRNA is directly involved in helping the scanning ribosome select the appropriate initiation codon on the mRNA through codon-anticodon base pa...

Yeast initiator tRNA identity elements cooperate to influence multiple steps of translation initiation

“…The A1:U72 base pair influences multiple steps of initiation Multiple studies, both in vivo andinvitro, have shown that the A1:U72 base pair in the acceptor stem of the initiator tRNAisacritical binding determinant for eIF2 (von PawelRammingen et al 1992;Astrom et al 1993;Farruggio et al 1996;D rabkin and RajBhandary 1998; Kapp and Lorsch 2004a). The results of our study indicate that, in addition to its key role in eIF2 d GTP d Met-tRNA i ternary complex formation, the A1:U72 base pair influences later steps in the pathway.…”

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…Substitution of the A1:U72 base pair in tRNA i Met by G1: C72, as found in tRNA e Met , impaired yeast cell growth (von Pawel-Rammingen et al 1992;Astrom et al 1993), binding of Met-tRNA i Met to eIF2 (Farruggio et al 1996;Kapp et al 2006), and TC binding to the 40S ribosome (Kapp et al 2006). Thus the identity of this base pair contributes both to TC formation and to later steps in the initiation pathway.…”

Twenty-first aminoacyl-tRNA synthetase–suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria

“…Total tRNA from yeast was isolated as described (26). The extent of aminoacylation was determined by fractionation of the tRNAs by acid-urea gel electrophoresis, followed by detection of the suppressor tRNAs by Northern blot hybridization with specific oligonucleotide probes (27).…”