title: A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs.
abstract: 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 mRNAs. The complex structure of these RNAs determined the correct positioning of the initiation codon in the ribosomal "P" site in binary complexes. Ribosomal binding and positioning on these mRNAs did not require the initiation factors eIF3, eIF4A, eIF4B, and eIF4F and translation of these mRNAs was not inhibited by a trans-dominant eIF4A mutant. Addition of Met-tRNAiMet, eIF2, and GTP to these binary ribosomal complexes resulted in formation of 48S preinitiation complexes. The striking similarities between this eukaryotic initiation mechanism and the mechanism of translation initiation in prokaryotes are discussed.
authors.affiliation: Department of Microbiology and Immunology, Morse Institute for Molecular Genetics, State University of New York Health Science Center at Brooklyn, Brooklyn, New York 11203, USA.
authors.forename: T V
authors.initials: TV
authors.lastname: Pestova
authors.forename: I N
authors.initials: IN
authors.lastname: Shatsky
authors.forename: S P
authors.initials: SP
authors.lastname: Fletcher
authors.forename: R J
authors.initials: RJ
authors.lastname: Jackson
authors.forename: C U
authors.initials: CU
authors.lastname: Hellen
mesh_terms: D000818:Animals; D001483:Base Sequence; D006692:Classical Swine Fever Virus / Q000235:genetics*; D018387:Codon, Initiator; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers; D005057:Eukaryotic Cells; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism / Q000494:pharmacology; D039621:Eukaryotic Initiation Factor-3; D039601:Eukaryotic Initiation Factor-4A; D039562:Eukaryotic Initiation Factor-4F; D039642:Eukaryotic Initiation Factors; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000378:metabolism / Q000494:pharmacology; D011387:Prokaryotic Cells; D014176:Protein Biosynthesis; D012358:RNA, Transfer, Met / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D011817:Rabbits; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D018387:Codon, Initiator; D017931:DNA Primers; D015852:Eukaryotic Initiation Factor-2; D039621:Eukaryotic Initiation Factor-3; D039562:Eukaryotic Initiation Factor-4F; D039642:Eukaryotic Initiation Factors; D010448:Peptide Initiation Factors; D012358:RNA, Transfer, Met; D012367:RNA, Viral; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins; C031799:eIF-4B; D039601:Eukaryotic Initiation Factor-4A