Abstract

Viral mRNA sequences with a type IV IRES are able to initiate translation without any host initiation factors. Initial recruitment of the small ribosomal subunit as well as two translocation steps before the first peptidyl transfer are essential for the initiation of translation by these mRNAs. Using electron cryomicroscopy (cryo-EM) we have structurally characterized at high resolution how the Cricket Paralysis Virus Internal Ribosomal Entry Site (CrPV-IRES) binds the small ribosomal subunit (40S) and the translocation intermediate stabilized by elongation factor 2 (eEF2). The CrPV-IRES restricts tvhe otherwise flexible 40S head to a conformation compatible with binding the large ribosomal subunit (60S). Once the 60S is recruited, the binary CrPV-IRES/80S complex oscillates between canonical and rotated states (Fernández et al., 2014; Koh et al., 2014), as seen for pre-translocation complexes with tRNAs. Elongation factor eEF2 with a GTP analog stabilizes the ribosome-IRES complex in a rotated state with an extra ~3 degrees of rotation. Key residues in domain IV of eEF2 interact with pseudoknot I (PKI) of the CrPV-IRES stabilizing it in a conformation reminiscent of a hybrid tRNA state. The structure explains how diphthamide, a eukaryotic and archaeal specific post-translational modification of a histidine residue of eEF2, is involved in translocation.

Highlights

  • During initiation of translation, a crucial step is the placement of the start codon and initiator tRNA in the P site of the ribosome (Schmeing and Ramakrishnan, 2009)

  • The first step in translation of an mRNA with a type IV IRES is the recruitment of the small ribosomal subunit (40S) (Jackson et al, 2010)

  • Type IV IRES sequences are able to fold independently into a compact structure in solution (Costantino and Kieft, 2005). Their tertiary structure allows crucial elements of the IRES to interact with the small ribosomal subunit via a cluster of specific ribosomal proteins (Fernandez et al, 2014)

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Summary

Introduction

A crucial step is the placement of the start codon and initiator tRNA in the P site of the ribosome (Schmeing and Ramakrishnan, 2009). One strategy employed by viruses relies on the use of structured sequences in their mRNA that allow them to bypass many or all aspects of canonical initiation (Pelletier and Sonenberg, 1988; Wilson et al, 2000; Kieft, 2009). These structured RNA sequences, termed IRES (for Internal Ribosomal Entry Site) elements, play a critical role in translation of their messages (Pelletier and Sonenberg, 1988; Jackson, 2005)

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