Fig. S4.

Reconstitution of translocation using eRF1. (Top) Bicistronic IRES RNAs were incubated with purified human ribosomes, GTP, and eukaryotic elongation factor 2, in the presence or absence of eRF1, as indicated. Ribosome positioning was monitored by primer extension analysis, and the resultant cDNA products were analyzed by denaturing polyacrylamide gel electrophoresis. Sequencing reactions are shown on the left, with the position of the +1 nucleotide as denoted. The locations of the major toeprints, including A6628 (+14), A6632 (+4 shift), and A6634 (+6 shift), are indicated. (Bottom) Schematics of IRES mutants and locations of the major toeprints.