(A) Sequences of −5U, +4U and MQQ-STOP mRNAs. The positions of Us (indicated by red asterisks) relative to the P-site codons (red arrows) in 80S initiation complexes or in pre-TCs are indicated. (B) Toe-printing analysis of 48S complexes and pre-TCs assembled on MQQ-STOP mRNA. The components of reaction mixtures are indicated. The position of ribosomal complexes are shown relative to the mRNA codon in the P site. Lanes C, T, A, G depict cDNA sequences corresponding to MQQ-STOP mRNA. (C) UV cross-linking of 32P-labeled −5U, +4U and MQQ-STOP mRNAs containing 4-ThioU with ribosomal proteins in recycled 40S subunit-containing complexes or in 80S initiation complexes, as indicated, assayed by SDS-PAGE and autoradiography. (D, E) Kinetics of [35S]MVHL tetrapeptide release in the presence (red circles) and in the absence (black circles) of eIFs at different ratios of eRFs and pre-TCs.