ABSTRACT
Translation initiation is a complex process in which initiator tRNA, 40S, and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of mRNA. The cap-binding complex eIF4F and the factors eIF4A and eIF4B are required for binding of 43S complexes (comprising a 40S subunit, eIF2/GTP/Met-tRNAi and eIF3) to the 5' end of capped mRNA but are not sufficient to promote ribosomal scanning to the initiation codon. eIF1A enhances the ability of eIF1 to dissociate aberrantly assembled complexes from mRNA, and these factors synergistically mediate 48S complex assembly at the initiation codon. Joining of 48S complexes to 60S subunits to form 80S ribosomes requires eIF5B, which has an essential ribosome-dependent GTPase activity and hydrolysis of eIF2-bound GTP induced by eIF5. Initiation on a few mRNAs is cap-independent and occurs instead by internal ribosomal entry. Encephalomyocarditis virus (EMCV) and hepatitis C virus epitomize distinct mechanisms of internal ribosomal entry site (IRES)-mediated initiation. The eIF4A and eIF4G subunits of eIF4F bind immediately upstream of the EMCV initiation codon and promote binding of 43S complexes. EMCV initiation does not involve scanning and does not require eIF1, eIF1A, and the eIF4E subunit of eIF4F. Initiation on some EMCV-like IRESs requires additional noncanonical initiation factors, which alter IRES conformation and promote binding of eIF4A/4G. Initiation on the hepatitis C virus IRES is even simpler: 43S complexes containing only eIF2 and eIF3 bind directly to the initiation codon as a result of specific interaction of the IRES and the 40S subunit.
Subject(s)
Globins/genetics , Peptide Chain Initiation, Translational , Ribosomes/metabolism , Amino Acid Sequence , Animals , Consensus Sequence , Eukaryotic Cells/physiology , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/metabolism , Humans , Molecular Sequence Data , Peptide Initiation Factors/chemistry , Peptide Initiation Factors/metabolism , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Transfer, Met/genetics , RNA, Transfer, Met/metabolism , Ribosomes/geneticsABSTRACT
The X-ray structure of the phylogenetically conserved middle portion of human eukaryotic initiation factor (eIF) 4GII has been determined at 2.4 A resolution, revealing a crescent-shaped domain consisting of ten alpha helices arranged as five HEAT repeats. Together with the ATP-dependent RNA helicase eIF4A, this HEAT domain suffices for 48S ribosomal complex formation with a picornaviral RNA internal ribosome entry site (IRES). Structure-based site-directed mutagenesis was used to identify two adjacent features on the surface of this essential component of the translation initiation machinery that, respectively, bind eIF4A and a picornaviral IRES. The structural and biochemical results provide mechanistic insights into both cap-dependent and cap-independent translation initiation.
Subject(s)
Peptide Initiation Factors/chemistry , Peptide Initiation Factors/genetics , Protein Biosynthesis/genetics , Binding Sites/genetics , Codon, Initiator/genetics , Conserved Sequence , Crystallography, X-Ray , Eukaryotic Initiation Factor-4G , Humans , Molecular Sequence Data , Mutagenesis/physiology , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino AcidABSTRACT
Mammalian eukaryotic initiation factor 4GI (eIF4GI) may be divided into three similarly sized regions. The central region (amino acids [aa] 613 to 1090) binds eIF3, eIF4A, and the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) and mediates initiation on this RNA. We identified the regions of eIF4GI that are responsible for its specific interaction with the IRES and that are required to mediate 48S complex formation on the IRES in vitro. Mutational analysis demarcated the IRES binding fragment of eIF4GI (aa 746 to 949) and indicated that it does not resemble an RNA recognition motif (RRM)-like domain. An additional amino-terminal sequence (aa 722 to 746) was required for binding eIF4A and for 48S complex formation. eIF4GI bound the EMCV IRES and beta-globin mRNA with similar affinities, but association with eIF4A increased its affinity for the EMCV IRES (but not beta-globin RNA) by 2 orders of magnitude. On the other hand, eIF4GI mutants with defects in binding eIF4A were defective in mediating 48S complex formation even if they bound the IRES normally. These data indicate that the eIF4G-eIF4A complex, rather than eIF4G alone, is required for specific high-affinity binding to the EMCV IRES and for internal ribosomal entry on this RNA.
Subject(s)
Encephalomyocarditis virus/genetics , Peptide Initiation Factors/genetics , Protein Biosynthesis , Animals , Binding Sites , Eukaryotic Initiation Factor-4A , Eukaryotic Initiation Factor-4G , Mutation , Peptide Initiation Factors/metabolism , Protein Binding , Ribosomal Proteins/genetics , Ribosomes/genetics , Ribosomes/metabolismABSTRACT
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.
Subject(s)
Peptide Chain Initiation, Translational , Peptide Initiation Factors/metabolism , Puromycin/analogs & derivatives , Ribosomes/metabolism , Amino Acid Sequence , Catalysis , Codon, Initiator , Eukaryotic Initiation Factor-1/metabolism , Eukaryotic Initiation Factor-2/metabolism , Eukaryotic Initiation Factor-3 , Eukaryotic Initiation Factor-5 , GTP Phosphohydrolases/metabolism , Guanosine Triphosphate/metabolism , Guanylyl Imidodiphosphate/metabolism , Humans , Hydrolysis , Molecular Sequence Data , Puromycin/biosynthesis , RNA, Messenger/metabolism , Recombinant Proteins/metabolismABSTRACT
Prolactin coding mRNA was shown to be a prevalent part of chum salmon (Oncorhynchus keta) pituitary poly(A)-RNA during the spawning period. Clone lambda gtPrk12 was selected from the pituitary cDNA library by means of hybridization with the prolactin probe, and a nucleotide sequence of the insertion was determined and compared to the prolactin coding sequences from rainbow trout and Pacific chinook salmon, which had been published earlier. The sequences compared exhibited a significant homology. The deduced amino acid sequence of the chum salmon prolactin differed from a sequence determined directly in a single position. The prolactin-coding sequence can be used for constructing the bacterial strain producing prolactin.
Subject(s)
DNA/genetics , Escherichia coli/genetics , Poly A/genetics , Prolactin/genetics , Protein Precursors/genetics , RNA/genetics , Amino Acid Sequence , Animals , Autoradiography , Base Sequence , Blotting, Northern , Cloning, Molecular , Electrophoresis, Agar Gel , Genes, Bacterial , Molecular Sequence Data , RNA, Messenger , Salmon , Sequence Homology, Nucleic AcidABSTRACT
The data are presented on the cloning and structural analysis of the cDNA coding for human prointerleukin-1 alpha and prointerleukin-1 beta (proIL-1 alpha and proIL-1 beta). The nucleotide sequences of proIL-1 alpha and proIL-1 beta cDNAs have been compared with the sequences published earlier. The nucleotide changes resulting in the aminoacid changes of the protein were not found. Some nucleotide changes were identified within the 3'-nontranslated region of the proIL-1 beta cDNA. The existence of the allelic variants for interleukin genes registered only on the gene level has been supposed.
