Binding Mode of the First Aminoacyl-tRNA in Translation Initiation Mediated by Plautia stali Intestine Virus Internal Ribosome Entry Site

Hiroshi Yamamoto,Nobuhiko Nakashima,Yuka Ikeda,Toshio Uchiumi

doi:10.1074/jbc.m610887200

Abstract

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.

Highlights

IntroductionIn the first elongation cycle, aminoacyl-tRNA should be brought to the A site with eukaryotic elongation factor 1A (eEF1A) and base-paired with the first decoded triplet at the 3Ј-border of PK I and translocated to the P site by the action of eEF2
By investigating the effect of the IRES binding on individual actions of eukaryotic elongation factor 1A (eEF1A) and eEF2, we have demonstrated that the first Gln-tRNA cannot bind efficiently through the action of eEF1A alone, but only bind efficiently upon addition of both eEF1A and eEF2 (Fig. 3A)
Because eEF2 alone does not function in Gln-tRNA binding at all, eEF1A should play an essential role in temporal binding of Gln-tRNA to the ribosomal A site and eEF2 efficiently translocating it to the P site

Summary

Introduction

In the first elongation cycle, aminoacyl-tRNA should be brought to the A site with eEF1A and base-paired with the first decoded triplet at the 3Ј-border of PK I and translocated to the P site by the action of eEF2. In usual translation the translocation is an event involving peptidyl-tRNA, not aminoacyl-tRNA [14] This process has been demonstrated by toeprinting assays in a system reconstituted with eEF1A, eEF2, ribosomes, aminoacyl-tRNA, and CrPV IRES [11, 12], it remains to be clarified how the IRES binding affects each activity of eEF1A or eEF2 on the ribosome. IRES markedly enhances chemical modification of bases in helix 18 of 18 S rRNA located at the A site These effects of IRES binding on ribosomal structure and function seem to be closely related to the unique process of IGR-IRES-mediated translation initiation

Methods

Results

Conclusion