Figure 1. Ski-mediated extraction of mRNA from ribosomal complexes.
(A) Purified Ski complex, analyzed by SDS-PAGE followed by fluorescent SYPRO staining. (B) Schematic representation of 80S IC, EC and pre-TC complexes. (C) Schematic representation of MF-Stop, MLLSSF-Stop and MVLL-Stop mRNAs, showing sequences of their 5’UTRs and coding regions. Upstream codons that are cognate or near-cognate to the P site tRNAs in the assembled ECs and on which ribosomes were halted during Ski-mediated mRNA extraction are in red and bracketed. (D) The activity of the Ski complex on 80S ICs and pre- and post-translocation ECs formed on MF-Stop mRNA, assayed by toe-printing. (E) The model for testing of the mechanism of Ski’s action. (F-G) The activity of the Ski complex on ECs formed on MLLSSF-Stop mRNA and containing MLLSS-tRNASer and the UCU codon in the P site, depending on (F) ATP and (G) free [Mg2+], assayed by toe-printing. Red arrows indicate ribosomal complexes halted at upstream codons (shown in red on the left). Separation of lanes in panels D and F by white lines indicates that they were juxtaposed from the same gel. (H) Elongation by ribosomes halted at the upstream codon during Ski-mediated mRNA extraction from pre-TCs formed on MVLL-Stop mRNA, assayed by toe-printing. Red arrows indicate ribosomal complexes formed at upstream codons (shown in red on the left) before and after one round of elongation. (I) The influence of eRF1•eRF3 and the Ski complex on ECs formed on MLLSSF-Stop mRNA and containing [35S]MLLSS-tRNALeu in the P site and the UUC(Phe) codon in the A site, depending on Saa-tRNA, eEF1H and eEF2, assayed by SDS-PAGE. The positions of peptidyl-tRNAs are indicated. (J, K) Dissociation of ECs in post- (J) and pre- (K) translocation states formed on MF-Stop mRNA using [32P]60S subunits by combinations of the Ski complex and Pelota/Hbs1/ABCE1 in the presence of eIF6, assayed by SDG centrifugation.