Structural characterization of ribosome recruitment and translocation by type IV IRES

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 the 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.DOI: http://dx.doi.org/10.7554/eLife.13567.001

Dynamics of IRES-mediated translation

“…eEF2 †GDPCP and eEF2-mediated ribosome and IRES conformational changes were observed [56]. eEF2 binding and GTPase activation occur via a conserved mechanism previously described for prokaryotic GTPases EF-G and EF-Tu [57].…”

“…In particular, during canonical translation, both the acceptor stem and peptidyl moiety are needed for frame maintenance during translocation [65,66], and their absence from PKI suggests that additional IRES elements are probably involved in frame selection during pseudotranslocation. In the cryo-EM reconstruction of 40S : CrPV IRES particles (3.8 Å ), the VRL density is fragmented, suggesting a partial ordering of the IRES element that is not apparent in 80S : CrPV IRES reconstructions [56]. One possibility is that transient interactions of the VRL with the 40S subunit facilitate events downstream of 80S recruitment [56].…”

“…eEF2 binding and GTPase activation occur via a conserved mechanism previously described for prokaryotic GTPases EF-G and EF-Tu [57]. Domains II and V of eEF2 anchor the GTPase to the ribosome through contacts with uS12, resulting in approximately 38 hyper-rotation of the 40S subunit and further outward displacement of the L1 stalk compared with the rotated state of the 80S : CrPV IRES [46,56]. PKI is displaced 10 Å towards the P site adopting an A/P hybrid-like conformation [56,58].…”

“…Domains II and V of eEF2 anchor the GTPase to the ribosome through contacts with uS12, resulting in approximately 38 hyper-rotation of the 40S subunit and further outward displacement of the L1 stalk compared with the rotated state of the 80S : CrPV IRES [46,56]. PKI is displaced 10 Å towards the P site adopting an A/P hybrid-like conformation [56,58]. Domain IV of eEF2 rests near the major groove of the ASL-like element of PKI, while eEF2 residues H694 and H699 interact directly with PKI.…”

“…To explore how eEF2 drives this pseudotranslocation step, the cryo-EM structure of eEF2 : 80S : CrPV IRES complex was determined at high resolution (3.6 Å ) [56]. eEF2 †GDPCP and eEF2-mediated ribosome and IRES conformational changes were observed [56].…”