A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initation of hepatitis C and classical swine fever virus RNAs
- Tatyana V. Pestova1,2,
- Ivan N. Shatsky2,
- Simon P. Fletcher3,
- Richard J. Jackson3, and
- Christopher U.T. Hellen1,4
- 1Department of Microbiology and Immunology, Morse Institute for Molecular Genetics, State University of New York Health Science Center at Brooklyn, Brooklyn, New York 11203; 2A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia; 3Department of Biochemistry, University of Cambridge, Cambridge, CB2 1QW, UK
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–tRNAi Met, 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.
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Footnotes
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↵4 Corresponding author.
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E-MAIL chellen{at}netmail.hscbklyn.edu; FAX (718) 270-2656.
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- Received June 12, 1997.
- Accepted October 29, 1997.
- Cold Spring Harbor Laboratory Press
