In cap-dependent translation initiation, the open reading frame (ORF) of mRNA is established by the placement of the AUG start codon and initiator tRNA in the ribosomal peptidyl (P) site. Internal ribosome entry sites (IRESs) promote translation of mRNAs in a cap-independent manner. We report two structures of the ribosome-bound Taura syndrome virus (TSV) IRES belonging to the family of Dicistroviridae intergenic IRESs. Intersubunit rotational states differ in these structures, suggesting that ribosome dynamics play a role in IRES translocation. Pseudoknot I of the IRES occupies the ribosomal decoding center at the aminoacyl (A) site in a manner resembling that of the tRNA anticodon-mRNA codon. The structures reveal that the TSV IRES initiates translation by a previously unseen mechanism, which is conceptually distinct from initiator tRNA-dependent mechanisms. Specifically, the ORF of the IRES-driven mRNA is established by the placement of the preceding tRNA-mRNA–like structure in the A site, whereas the 40S P site remains unoccupied during this initial step.Protein synthesis relies on precise placement of the ORF within the ribosome during translation initiation. Canonical initiation in eukaryotes depends on a 7-methylguanosine cap at the 5′ terminus of mRNA and on extraribosomal initiation factors (
1). Following a stepwise assembly, the 80S initiation complex contains the initiator methionyl-tRNA
Met and the AUG start codon in the peptidyl (P) site. Some viral mRNAs use alternative cap-independent mechanisms that involve internal ribosome entry sites (IRESs) (
2). IRESs are folded RNA structures in the 5′ UTR that promote formation of the 80S initiation complex in the presence of fewer initiation factors than required for cap-dependent initiation (
3).The ribosomal P-site employment in initiation is thought to be ubiquitous for cap-dependent and IRES-dependent translation (
4). Of the four groups of known IRESs, the most streamlined mechanism has been described for IRESs from the Dicistroviridae family of arthropod-infecting viruses. The Dicistroviridae genome has two ORFs separated by an intergenic region (IGR). The IGR contains an IRES that drives translation of the second ORF without the aid of initiation factors (
4). Based on phylogenetic analyses of the structural polyprotein ORF2 and IGR IRES, the Dicistroviridae viruses are divided into the genus
Cripavirus [including cricket paralysis virus (CrPV), Drosophila C virus, and
Plautia stali intestine virus (PSIV)] and
Aparavirus [including Taura syndrome virus (TSV), Kashmir bee virus, and acute bee paralysis virus] (
4). Biochemical studies suggest that despite differences between some secondary structure elements of
Cripavirus and
Aparavirus IRESs, the molecular mechanisms of translation initiation are similar (
5). IGR IRESs can initiate translation on ribosomes from yeast, wheat, human, and other eukaryotic organisms, indicating that the molecular mechanism of IGR IRES-driven initiation in eukaryotes is conserved and is not species-specific (
6–
10).In contrast to cap-dependent initiation and initiation from other groups of IRESs, translation from IGR IRESs starts from a non-AUG start codon and does not involve initiator methionyl-tRNA
Met. Translation from the majority of IGR IRESs, including the CrPV and TSV IRESs, initiates with alanyl-tRNA
Ala (
7,
9,
10). IGR IRESs contain three pseudoknots. At the 5′ region, pseudoknot II (PKII) and PKIII, which are critical for formation of the 40S•IRES and 80S•IRES complexes (
8,
9), form a double-nested pseudoknot (
11,
12). PKI, located immediately upstream of the start codon, forms a separate domain at the 3′ region of the IRES. This domain is essential for the function of IGR IRESs (
13). The crystal structure of an isolated PKI of the CrPV IGR IRES shows that the pseudoknot resembles the anticodon stem loop of tRNA bound to a cognate mRNA codon (
14,
15). Isolated PKI of CrPV and PSIV IRESs binds to the P site of the bacterial 70S ribosome, demonstrating that PKI has an affinity to the highly conserved tRNA binding sites on the ribosome (
16).The molecular mechanism of translation initiation by IGR IRESs is not fully understood. The current view is that upon formation of the 80S•IRES complex, the PKI is placed in the P site on the small subunit, in a manner mimicking the initiator methionyl-tRNA
Met and the AUG codon (
6–
8,
10,
17). In this mode, the IRES would position the ORF on the ribosome by presenting the initiating alanine codon in the A (aminoacyl) site. The structural studies of the mechanism, however, have been inconclusive. Previous electron cryomicroscopy (cryo-EM) reconstruction of the CrPV IRES bound to human ribosomal 40S subunit revealed the IRES density spanning from the A site to beyond the exit (E) site (
18). The interpretation of the 40S•IRES map favored a model in which PKI interacts with the P-site region (
18), although the 20-Å map lacked detailed features in this location. Cryo-EM studies of the 80S ribosome-bound CrPV IRES suggested that upon subunit joining and 80S•IRES complex formation, the IRES may rearrange relative to the 40S subunit (
18), and/or reposition PKI in the vicinity of the A and P sites, yet potentially present the downstream alanine codon in the A site (
19). The density for the PKI region in these 20-Å and 7.3-Å cryo-EM reconstructions was, however, significantly weaker than that for the rest of the CrPV IRES (
18,
19), and it remained unclear how the IGR IRESs initiate translation by accurately positioning the ORF on the 80S ribosome. We report here ∼6-Å cryo-EM structures of the initiation 80S complex bound with an intergenic IRES, which provide structural insights into the mechanism of IGR IRES-driven initiation.
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