Binding mode of the first aminoacyl-tRNA in translation initiation mediated by Plautia stali intestine virus internal ribosome entry site.
Eukaryotic ribosomes directly bind to the intergenic region-internal ribosome entry site (IGR-IRES) of Plautia stali intestine virus (PSIV) and initiate translation without either initiation factors or initiator Met-tRNA. We have investigated the mode of binding of the first aminoacyl-tRNA in translation initiation mediated by the IGR-IRES. Binding ability of aminoacyl-tRNA to the first codon within the IGR-IRES/80 S ribosome complex was very low in the presence of eukaryotic elongation factor 1A (eEF1A) alone but markedly enhanced by the translocase eEF2. Moreover, eEF2-dependent GTPase activity of the IRES/80 S ribosome complex was 3-fold higher than that of the free 80 S ribosome. This activation was suppressed by addition of the antibiotics pactamycin and hygromycin B, which are inhibitors of translocation. The results suggest that translocation by the action of eEF2 is essential for stable tRNA binding to the first codon of the PSIV-IRES in the ribosome. Chemical probing analysis showed that IRES binding causes a conformational change in helix 18 of 18 S rRNA at the A site such that IRES destabilizes the conserved pseudoknot within the helix. This conformational change caused by the PSIV-IRES may be responsible for the activation of eEF2 action and stimulation of the first tRNA binding to the P site without initiation factors.
Yamamoto
Hiroshi
H
Department of Biology, Faculty of Science, Niigata University, Niigata 950-2181, Japan.
Nakashima
Nobuhiko
N
Ikeda
Yuka
Y
Uchiumi
Toshio
T
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D015977:Eukaryotic Initiation Factor-1 / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D020062:Heteroptera / Q000821:virology*; D007304:Insect Viruses / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020652:Peptide Elongation Factor 2 / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012343:RNA, Transfer / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry*
D000900:Anti-Bacterial Agents; D015977:Eukaryotic Initiation Factor-1; D020652:Peptide Elongation Factor 2; C026022:eukaryotic peptide initiation factor-1A; D006153:Guanosine Diphosphate; D012343:RNA, Transfer