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. 2017 Sep 12;36(20):2968–2986. doi: 10.15252/embj.201797079

Figure 1. UPF3B delays translation termination in vitro .

Figure 1

  1. Scheme of the MVHC‐STOP mRNA.
  2. Toeprinting analysis of ribosomal complexes obtained by incubating pre‐TCs assembled on MVHC‐STOP mRNA (MVHC‐pre‐TCs) with UPF1, UPF2L, UPF3B or BSA at 1 mM free Mg2+ followed by termination with limiting amounts of eRF1 and eRF3a. The positions of pre‐TCs, post‐TCs and full‐length cDNA are indicated. In all toeprinting analyses the box marked TAA indicates the stop codon in the cDNA sequence corresponding to the MVHC‐STOP mRNA. Asterisks mark initiation and elongation complexes in all toeprinting analyses. Representative of five independent experiments. Lanes C, T, A, G indicates the sequence of the cDNA derived from the MVHC‐STOP mRNA using the same primer as the toeprint experiment. The box ‘TAA’ indicates the position of the stop codon.
  3. Kinetics of [35S]‐peptidyl‐tRNA hydrolysis in the presence of eRF1 and eRF3a (black circles) or eRF1, eRF3a and UPF3B (white triangles). Termination reactions were assembled as in (B). A value equal to 1 corresponds to the maximum value for peptide release triggered by eRF1 and eRF3a. Data points show the mean of three experiments ± SEM.
  4. UPF1 in vitro phosphorylation by SMG1‐8‐9 in the presence of UPF2L and/or UPF3B and in the presence (lanes 7–12) or absence (lanes 1–6) of the eRFs. In lanes 13–16 UPF2L and/or UPF3B were added after UPF1 phosphorylation. Samples were analysed by SDS–PAGE, Coomassie‐stained to control for equal loading (lower panel) and autoradiographed (upper panel). SMG1 autophosphorylation (P‐SMG1) confirms equal SMG1‐activity in all samples. UPF1 is represented by the lower and UPF2L by the upper of the two closely migrating bands between 125 and 130 kDa in the Coomassie‐stained gel. Representative of two independent experiments.
  5. Toeprinting analysis of ribosomal complexes obtained by incubating pre‐TCs with UPF1, UPF2L, UPF3B, SMG1‐8‐9 or BSA at 1 mM free Mg2+ as indicated followed by translation termination by eRF1 and eRF3a. See also Fig EV2. Representative of three independent experiments.

Source data are available online for this figure.