Since the reinitiation mechanism shows a fairly strong preference for an AUG codon, it seems a certainty that initiator Met-tRNA is involved. This, in turn, implies that the ribosome lanes of the left panel show translations of mutant mRNAs in which the codon -1 of the AUG start codon and the start codon (+1) were mutated to the indicated codons in the context of the BM2start +63 RNA. Lane numbers are shown below the figure. Right panel: Translations of RNAs containing stop and start codons at the indicated positions. C) Capped BMV (BMV) RNAs, BM2 wt reporters (Cap-wt), CrPV-IRES driven BM2wt (CrPV-wt) and BM2start +63 (CrPV+63) RNAs were translated in control (C) and eIF4G-depleted (D) RRLs in the absence or presence of ,1 mM dominant-negative eIFA (4A-R362Q). RNAs were translated as described in [26] and analysed as above. doi:10.1371/journal.pone.0016822.g006 which has just terminated at the stop codon must dissociate into subunits prior to reinitiation, in order to allow eIF2/GTP/Met-tRNA i ternary complex binding to the 40S subunit. So it is a 40S subunit rather than an 80S ribosome that interacts with TURBS motif 1. The fact that reinitiation efficiency decreases quite abruptly with increasing distance of displacement of the stop [11] ) is shown with motif 1 shown in red, the termination-reinitiation overlap shown in cyan and the sequences likely to be occupied by a ribosome terminating at the M1 ORF are 39 of the purple line. Mutations were introduced at the bottom of stem 2, with both the 2 nt and 3 nt substitution mutations shown next to arm 1 (A1) or arm 2 (A2). Messenger RNAs derived from these plasmids and control mRNA (204, ps) were translated and analysed as detailed in the legend to Figure 1 . RRF denotes the relative reinitiation frequency adjusted for relative methionine content as compared to the wt (set as 100%). (B) The mfold 2 structure (from [11] ) is shown with motif 1, the termination-reinitiation overlap, and the ribosome protected region marked as above. Mutations were introduced at the bottom of stem 29 and are shown next to arm 1 (A19) or arm 2 (A29). Messenger RNAs derived from these plasmids and a control mRNA (204) were translated and analysed as above. RRF denotes the relative reinitiation frequency adjusted for relative methionine content as compared to the wt (set as 100%). doi:10.1371/journal.pone.0016822.g008 codon further downstream ( Figure 5 ) favours a model in which this 40S subunit is transferred directly from the termination site to the TURBS, rather than dissociating from the mRNA after termination followed by reassociation with the TURBS.