FIGURE 4.

(A) RelE printing and toeprinting of HRV-Fluc and FMDV-Fluc RNAs in the combined RRL-HeLa extracts. cDNA ladders for these RNAs are shown on the left and right of the gel, respectively. Groups of lanes marked “extension” show the results of toeprinting, whereas those denoted as “deproteinization extension” correspond to RelE-print assays. For comparison, the assays with RRL only also presented (lanes 1,2,4,5,8,9,11,12). Lanes 1 and 8 are control assays where the reconstitution was blocked by 30 mM of magnesium. (B) RelE-induced cleavage of the β-globin mRNA in 48S complexes formed on near-cognate codons in the absence of eIF1. The translation initiation complexes on the β-globin mRNA were assembled under the same conditions and with the same batches of components as in the experiments shown in Fig. 2 except eIF1 was not added to the reconstitution system. For higher resolution of upper bands, a long run gel was required (2 h 10 min instead of regular 1 h 10 min), and therefore, the coding part of the mRNA is not presented in the gel. The positions of toeprint and RelE-print bands as well as the sequence of the β-globin mRNA are shown on the right of the gel. The sequence of the β-globin mRNA is also presented on the right of the autograph. Lane 1 shows results of toeprinting in the absence of eIF1. Lanes 2 and 3 are the primer extensions on free mRNAs isolated from reconstitution mixtures treated (+) and untreated (−) with RelE. The position of AUG triplet and near-cognate triplets are shown in boxes. Empty arrows show the positions of RelE attack. Asterisks denote the position of the initiation codon.