Volume 51, Issue 2, Pages 249-264 (July 2013) Reinitiation and Other Unconventional Posttermination Events during Eukaryotic Translation  Maxim A. Skabkin, Olga V. Skabkina, Christopher U.T. Hellen, Tatyana V. Pestova  Molecular Cell  Volume 51, Issue 2, Pages 249-264 (July 2013) DOI: 10.1016/j.molcel.2013.05.026 Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Mammalian Posttermination Ribosomes Can Migrate to Nearby Codons that Are Cognate to the P Site Deacylated tRNA (A) Structure of MVHL-STOP, MVHC-STOP, and MVHC-STOP/ss3′ mRNAs. Upstream and downstream codons that are identical to the last codon of the encoded tetrapeptide are in red and underlined. (B–F) Toeprinting analysis of ribosomal complexes obtained by treating pre-TCs formed on MVHL-STOP mRNA (B), MVHC-STOP mRNA (C), MVHC-STOP/ss3′ mRNA (D), MVHL-STOP mRNAs containing different stop codons (E), or MVKC-STOP mRNA (F) with eRF1, eRF3, and puromycin at the indicated free [Mg2+]. Black arrows show the positions of pre-TCs, post-TCs, and recycled 40S subunits. Red arrows indicate the positions of migrated posttermination ribosomes. Upstream and downstream codons that are identical to the last codon of the encoded tetrapeptide are in red. The upper panel of (F) shows the structure of MVKC-STOP mRNA. (G and H) Toeprinting analysis of ribosomal complexes obtained by treating pre-TCs formed on MVHL-STOP mRNA (G) and MVHC-STOP mRNA (H) with eRF1, eRF3, or puromycin in the presence or absence of eIF3, eIF1, and eIF1A at the indicated free [Mg2+]. Black arrows show the positions of pre-TCs, post-TCs, and recycled 40S subunits. Red arrows indicate the positions of migrated posttermination ribosomes. Upstream codons that are identical to the last codon of the encoded tetrapeptide are in red. (I) Primer-extension analysis of A site mRNA cleavage induced by RelE in pre- and post-TCs assembled on MVHC-STOP mRNA. The positions of cleavages and of corresponding P site codons are indicated. (J) Toeprinting analysis of ribosomal complexes obtained by treating pre-TCs formed on MVHL-STOP (left panel) and MVHC-STOP (right panel) with eRF1 and eRF3 in the presence or absence of ABCE1 at the indicated free [Mg2+]. Black arrows show the positions of pre-TCs, post-TCs, and recycled 40S subunits. Red arrows indicate the positions of migrated posttermination ribosomes. Upstream codons that are identical to the last codon of the encoded tetrapeptide are in red. Molecular Cell 2013 51, 249-264DOI: (10.1016/j.molcel.2013.05.026) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 The Ability of Posttermination Ribosomes with a Vacant P Site to Rebind tRNAs (A–C) Toeprinting analysis of ribosomal complexes obtained by treating pre-TCs formed on MVHC-STOP mRNA with eRF1⋅eRF3 in the presence or absence of tRNAiMet (A), with puromycin in the presence or absence of tRNAiMet (B), or with eRF1, eRF1⋅eRF3, or puromycin in the presence or absence of tRNAHis or tRNALys (C) at the indicated free [Mg2+]. Red arrows show the positions of ribosomal complexes arrested at AUG (Met), CAC (His), or AAA (Lys) codons. Molecular Cell 2013 51, 249-264DOI: (10.1016/j.molcel.2013.05.026) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 eEF2 Destabilizes Post-TCs and Promotes Ribosome Migration (A–E) Toeprinting analysis of ribosomal complexes obtained by treating pre-TCs formed on MVHC-STOP mRNA (A), MVHL-STOP mRNA (B), MVKC-STOP mRNA (C), MVHC-STOP mRNA (D), and MVHL-STOP/C-less mRNAs (E) with combinations of eRF1, eRF3, puromycin, eRF1(AGQ), and eEF2 at the indicated free [Mg2+]. Black arrows show the positions of pre- and post-TCs. Red arrows indicate the positions of migrated posttermination ribosomes. Upstream codons that are identical to the last codon of the encoded tetrapeptide are shown in red. Molecular Cell 2013 51, 249-264DOI: (10.1016/j.molcel.2013.05.026) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 The E Site tRNA Increases the Stability of Post-TCs (A–C) Toeprinting analysis of ribosomal complexes obtained by treating pre-TCs formed on MVKC-STOP mRNA (A, left panel), MVHC-STOP mRNA (A, right panel), MVHL-STOP mRNA (B), and MVHC-STOP mRNA (C) with combinations of eRF1, eRF3, puromycin, eEF2, and ABCE1 in the presence or absence of tRNALys at the indicated free [Mg2+]. Black arrows show the positions of pre- and post-TCs. Red arrows indicate the positions of migrated posttermination ribosomes. Upstream codons that are identical to the last codon of the encoded tetrapeptide are shown in red. Molecular Cell 2013 51, 249-264DOI: (10.1016/j.molcel.2013.05.026) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 Reinitiation following ABCE1-Mediated Recycling (A) Structure of Stem-MVHL-STOP mRNA. (B) Toeprinting analysis of 48S complex formation on Stem-MVHL-STOP mRNA in the presence of combinations of eIFs as indicated. (C–J) Toeprinting analysis of ribosomal complexes obtained by treating pre-TCs formed on Stem-MVHL-STOP mRNA with eRF1⋅eRF3 in the presence or absence of different combinations of ABCE1, Met-tRNAiMet, eIF2, eIF3, eIF1, eIF1A, eIF3j, eIF4A, eIF4B, eIF4F, eIF4G, Ligatin, and excess competitor (CAA)n-GUS mRNA at the indicated free [Mg2+] (C, D, E, F, G, H, I, and J). The positions of pre-TCs, post-TCs, and tRNA- and mRNA-associated recycled 40S subunits are indicated by black arrows. The positions of 48S complexes formed on AUG triplets upstream and downstream of the stop codon are shown in red and blue, respectively. Molecular Cell 2013 51, 249-264DOI: (10.1016/j.molcel.2013.05.026) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 Reinitiation following eIF-Mediated Recycling (A–D) Toeprinting analysis of ribosomal complexes obtained by treating pre-TCs formed on Stem-MVHL-STOP mRNA with eRF1, eRF1⋅eRF3, or puromycin in the presence or absence of different combinations of ABCE1, Met-tRNAiMet, and eIF2, eIF3, eIF1, eIF1A, eIF3j, eIF4A, eIF4B, eIF4F, and eIF4G at 1.5 mM Mg2+ (A), various indicated [Mg2+] (B), 1 mM Mg2+ (C), and various indicated [Mg2+] (D). The positions of pre- and post-TCs are indicated by black arrows. The positions of 48S complexes formed at the AUG triplets upstream and downstream of the stop codon are shown in red and blue, respectively. (E–G) Dissociation of pre-TCs, assembled on Stem-MVHL-STOP mRNA with 32P-labeled 60S subunits, after incubation with combinations of eRFs, ABCE1, and eIFs at 1 mM Mg2+ (E), 2.5 mM Mg2+ (F), and 1 mM Mg2+ (G), assayed by SDG centrifugation. Upper fractions were omitted for clarity. Molecular Cell 2013 51, 249-264DOI: (10.1016/j.molcel.2013.05.026) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 7 Reinitiation on mRNA with an Unstructured Region Downstream of the Stop Codon (A) Structure of Stem-MVHC/ss3′ mRNA. (B–D) Toeprinting analysis of ribosomal complexes obtained by treating pre-TCs formed on Stem-MVHC/ss3′ mRNA with combinations of eRF1⋅eRF3 in the presence or absence of different combinations of ABCE1, Met-tRNAiMet, and eIF2, eIF3, eIF1, eIF1A, eIF4A, eIF4B, eIF4F, and eIF4G at 2.5 mM Mg2+ (B), 1 mM Mg2+ (C), and 1 mM Mg2+ (D). The positions of pre-TCs, post-TCs, and tRNA- and mRNA-associated recycled 40S subunits are indicated by black arrows. The positions of 48S complexes formed at the AUG triplets upstream and downstream of the stop codon are shown in red and blue, respectively. (E and F) Toeprinting analysis of ribosomal complexes obtained by treating pre-TCs formed on MVHL-STOP mRNA with eRF1⋅eRF3 in the presence or absence of different combinations of ABCE1, Met-tRNAiMet, tRNAiMet, eIF2, eIF3, eIF1, eIF1A, Ligatin, eEFs, 60S subunits, and Val-tRNAVal at 2.5 mM Mg2+ (E and F). The positions of pre-TCs, post-TCs, and tRNA- and mRNA-associated recycled 40S subunits are indicated by black arrows. The positions of 48S complexes formed at the upstream AUG triplet and to elongation complexes formed at the GUG codon are shown in red and green, respectively. Molecular Cell 2013 51, 249-264DOI: (10.1016/j.molcel.2013.05.026) Copyright © 2013 Elsevier Inc. Terms and Conditions