Two additional regions of the IGR-IRES are functionally relevant: the L1.1 region, responsible for the interaction with the L1 stalk of the large ribosomal subunit and PKI. As a result, two stem loops (SL-IV and SL-V) are exposed and are responsible for the bulk of interactions with the small ribosomal subunit ( Fernández et al., 2014 Schüler et al., 2006 Spahn et al., 2004a). Two of these pseudoknots (PKII and PKIII) and accessory sequences, fold into a compact domain following a scheme also seen in other structured RNAs ( Kieft, 2009 Au et al., 2015). The approximately 190 nucleotide sequence folds into a compact, well-defined three-dimensional structure making use of three internal pseudoknots (PKI-III, Figure 1) ( Costantino and Kieft, 2005 Costantino et al., 2008 Pestova et al., 2004 Schüler et al., 2006 Pestova and Hellen, 2003). Extensive biochemical and structural characterization of these IRES sequences has provided a detailed picture on how they work at a molecular level. These IRESs are typically found in the intergenic region (IGR) of a family of + sense RNA viruses called Dicistroviridae hence they are also known as IGR-IRES ( Wilson et al., 2000 Sasaki and Nakashima, 1999). Type IV IRESs form a homogenous class of viral RNAs, which completely dispense with the requirement for any IFs and thus are able to directly recruit and assemble a translating 80S ribosome by themselves. Viral IRES sequences can be classified according to their level of dependence on IFs ( Filbin and 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). 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). However, viruses, which rely on the host machinery to translate their genomes, often target initiation to hijack host ribosomes ( Jackson, 2005 Walsh and Mohr, 2011). The complexity of initiation in eukaryotes is used by cells to regulate translation ( Jackson et al., 2010). Subsequently, the resulting 48S complex scans along the mRNA to reach the start codon ( Aitken and Lorsch, 2012 Hinnebusch, 2014). A subset of IFs bind to the capped 5’ end of a mRNA followed by recruitment of a 43S complex of the small subunit (with additional IFs). In eukaryotes, initiation involves almost a dozen protein initiation factors (IFs), several of which are large multi-subunit complexes. The structure explains how diphthamide, a eukaryotic and archaeal specific post-translational modification of a histidine residue of eEF2, is involved in translocation.ĭuring 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). 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. Elongation factor eEF2 with a GTP analog stabilizes the ribosome-IRES complex in a rotated state with an extra ~3 degrees of rotation. 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. The CrPV-IRES restricts the otherwise flexible 40S head to a conformation compatible with binding the large ribosomal subunit (60S). 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). 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. Viral mRNA sequences with a type IV IRES are able to initiate translation without any host initiation factors.
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