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Initiation of translation of hepatitis C virus and classical swine fever virus mRNAs results from internal ribosomal entry. We reconstituted internal ribosomal entry in vitro from purified translation components and monitored assembly of 48S ribosomal preinitiation complexes by toe-printing. Ribosomal subunits (40S) formed stable binary complexes on both(More)
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(More)
Eukaryotic translation initiation begins with assembly of a 43S preinitiation complex. First, methionylated initiator methionine transfer RNA (Met-tRNAi(Met)), eukaryotic initiation factor (eIF) 2, and guanosine triphosphate form a ternary complex (TC). The TC, eIF3, eIF1, and eIF1A cooperatively bind to the 40S subunit, yielding the 43S preinitiation(More)
The scanning model of translation initiation is a coherent description of how eukaryotic ribosomes reach the initiation codon after being recruited to the capped 5' end of messenger RNA. Five eukaryotic initiation factors (eIF 2, 3, 4A, 4B and 4F) with established functions have been assumed to be sufficient to mediate this process. Here we report that eIF1(More)
Positioning of the translation initiation complex on mRNAs requires interaction between the anticodon of initiator Met-tRNA, associated with eIF2-GTP and 40S ribosomal subunit, and the cognate start codon of the mRNA. We show that an internal ribosome entry site located in the genome of cricket paralysis virus can form 80S ribosomes without initiator(More)
Translation of picornavirus RNA is initiated after ribosomal binding to an internal ribosomal entry site (IRES) within the 5' untranslated region. We have reconstituted IRES-mediated initiation on encephalomyocarditis virus RNA from purified components and used primer extension analysis to confirm the fidelity of 48S preinitiation complex formation.(More)
Eukaryotic protein synthesis begins with assembly of 48S initiation complexes at the initiation codon of mRNA, which requires at least seven initiation factors (eIFs). First, 43S preinitiation complexes comprising 40S ribosomal subunits, eIFs 3, 2, 1, and 1A, and tRNA(Met)(i) attach to the 5'-proximal region of mRNA and then scan along the 5' untranslated(More)
After translational termination, mRNA and P site deacylated tRNA remain associated with ribosomes in posttermination complexes (post-TCs), which must therefore be recycled by releasing mRNA and deacylated tRNA and by dissociating ribosomes into subunits. Recycling of bacterial post-TCs requires elongation factor EF-G and a ribosome recycling factor RRF.(More)
Translation of hepatitis C virus (HCV) and classical swine fever virus (CSFV) RNAs is initiated by cap-independent attachment (internal entry) of ribosomes to the approximately 350-nucleotide internal ribosomal entry segment (IRES) at the 5' end of both RNAs. Eukaryotic initiation factor 3 (eIF3) binds specifically to HCV and CSFV IRESs and plays an(More)
Eukaryotic translation termination is triggered by peptide release factors eRF1 and eRF3. Whereas eRF1 recognizes all three termination codons and induces hydrolysis of peptidyl tRNA, eRF3's function remains obscure. Here, we reconstituted all steps of eukaryotic translation in vitro using purified ribosomal subunits; initiation, elongation, and termination(More)