guanosine-triphosphate and methionylpuromycin

All three kingdoms of life employ two methionine tRNAs, one for translation initiation and the other for insertion of methionines at internal positions within growing polypeptide chains. We have used a reconstituted yeast translation initiation system to explore the interactions of the initiator tRNA with the translation initiation machinery. Our data indicate that in addition to its previously characterized role in binding of the initiator tRNA to eukaryotic initiation factor 2 (eIF2), the initiator-specific A1:U72 base pair at the top of the acceptor stem is important for the binding of the eIF2.GTP.Met-tRNA(i) ternary complex to the 40S ribosomal subunit. We have also shown that the initiator-specific G:C base pairs in the anticodon stem of the initiator tRNA are required for the strong thermodynamic coupling between binding of the ternary complex and mRNA to the ribosome. This coupling reflects interactions that occur within the complex upon recognition of the start codon, suggesting that these initiator-specific G:C pairs influence this step. The effect of these anticodon stem identity elements is influenced by bases in the T loop of the tRNA, suggesting that conformational coupling between the D-loop-T-loop substructure and the anticodon stem of the initiator tRNA may occur during AUG codon selection in the ribosomal P-site, similar to the conformational coupling that occurs in A-site tRNAs engaged in mRNA decoding during the elongation phase of protein synthesis.

Topics: Base Sequence; Conserved Sequence; Eukaryotic Initiation Factor-1; Eukaryotic Initiation Factor-2; Eukaryotic Initiation Factor-5; Eukaryotic Initiation Factors; Guanosine Triphosphate; Molecular Sequence Data; Mutation; Nucleic Acid Conformation; Peptide Chain Initiation, Translational; Protein Biosynthesis; Protein Structure, Tertiary; Puromycin; Ribosomes; RNA, Fungal; RNA, Transfer, Met; Saccharomyces cerevisiae

Initiation of eukaryotic protein synthesis begins with the ribosome separated into its 40S and 60S subunits. The 40S subunit first binds eukaryotic initiation factor (eIF) 3 and an eIF2-GTP-initiator transfer RNA ternary complex. The resulting complex requires eIF1, eIF1A, eIF4A, eIF4B and eIF4F to bind to a messenger RNA and to scan to the initiation codon. eIF5 stimulates hydrolysis of eIF2-bound GTP and eIF2 is released from the 48S complex formed at the initiation codon before it is joined by a 60S subunit to form an active 80S ribosome. Here we show that hydrolysis of eIF2-bound GTP induced by eIF5 in 48S complexes is necessary but not sufficient for the subunits to join. A second factor termed eIF5B (relative molecular mass 175,000) is essential for this process. It is a homologue of the prokaryotic initiation factor IF2 (re and, like it, mediates joining of subunits and has a ribosome-dependent GTPase activity that is essential for its function.

Topics: Amino Acid Sequence; Catalysis; Codon, Initiator; Eukaryotic Initiation Factor-1; Eukaryotic Initiation Factor-2; Eukaryotic Initiation Factor-3; Eukaryotic Initiation Factor-5; GTP Phosphohydrolases; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Humans; Hydrolysis; Molecular Sequence Data; Peptide Chain Initiation, Translational; Peptide Initiation Factors; Puromycin; Recombinant Proteins; Ribosomes; RNA, Messenger

The rate of initiation of protein synthesis appears to be controlled at the level of recycling of eIF-2. In this process a new factor, designated eRF, plays an important role. The factor has been purified from the post-ribosomal supernatant and has been called formerly anti-HRI and anti-inhibitor [Amesz, H., Goumans, H., Haubrich-Morree, Th., Voorma, H.O., and Benne, R. (1979) Eur. J. Biochem. 98, 513-520]. Its effect on the initiation of protein synthesis has been studied in several assays: a small but distinct effect is found in the assay for the formation of a ternary complex between eIF-2, GTP and Met-tRNA; a 4-5-fold stimulation is obtained in assays for 40S preinitiation complex formation and in the methionyl-puromycin reaction. In the latter assay a catalytic use of eIF-2 occurs provided that eRF is present. eRF forms a complex with eIF-2 which results in a decrease of the affinity of eIF-2 for GDP, giving it the properties of a GDP/GTP exchange factor. The model stresses the catalytic use of eIF-2 in initiation provided that conditions are met for GDP/GTP exchange by a transient complex formation between eIF-2 and eRF. On the other hand, it is shown that phosphorylation of eIF-2 by the hemin-regulated inhibitor (HRI) abolishes the recycling of eIF-2, by the formation of another stable complex comprising eIF-2 alpha P, GDP and eRF.

Topics: Animals; Autoradiography; Catalysis; Chemical Phenomena; Chemistry; eIF-2 Kinase; Electrophoresis, Polyacrylamide Gel; Eukaryotic Initiation Factor-2; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Peptide Chain Initiation, Translational; Peptide Initiation Factors; Protein Binding; Protein Biosynthesis; Protein Serine-Threonine Kinases; Proteins; Puromycin; Rabbits; Reticulocytes; RNA, Transfer, Amino Acyl

Topics: Animals; Chromatography, Affinity; Eukaryotic Initiation Factor-2; Guanosine Triphosphate; Heme; Kinetics; Magnesium; Peptide Initiation Factors; Phosphorylation; Plants; Protein Biosynthesis; Proteins; Puromycin; Rabbits; Reticulocytes; Species Specificity; Triticum