We first demonstrated that under simple experimental conditions (where tRNA was non-enzymatically delivered at the ribosomal A site), toeprinting allows localization of the ribosome on the mRNA containing the wild-type HIV-1 slippery sequence (mSP-HIV-1) but not the downstream hairpin. The sequences and secondary structures of the mRNA constructs used are derived from T4 gene 32 mRNA in which we introduced the HIV-1 frameshifting signals as shown in Figure 1 . In the case of the mSP-HIV-1 construct, two distinct toeprints are observed at +15 and +16 when tRNA Phe is bound at the P site ( Figure S1A , lane F), in correspondence with the two possible reading frames created by the slippery sequence. A third toeprint at +17 appears when tRNA Leu subsequently binds at the A site ( Figure S1A , lane F 0 ). This is thought to be due to a conformational change in the ribosomal complex following A-site binding so that a single position of the tRNA in the A site results in a doublet of bands (at positions +16 and +17) (46, 47) . It is important to note that the signal at +15 did decrease, indicating that binding of the tRNA Leu at the A site assisted in the positioning of the mRNA with tRNA Phe preferentially bound to the u +1 u +2 u +3 codon adjacent to the leucine codon u +4 u +5 a +6 . After EF-G catalyzed translocation of tRNA Leu to the P site ( Figure S1A , lane L), the toeprints at +16, +17 were moved to +20 and +21, corresponding to a 4-nt translocation event. We cannot conclude if a toeprint at +19 exists since this band is also present in the control lane (without ribosome). However, if it exists, this toeprint is very weak. When tRNA Gly is subsequently added to the EF-G containing reaction mixture ( Figure S1A , lane G), translocation proceeds further giving the expected toeprints at +22 and +23 for tRNA Gly bound to P site and paired with the g +7 g +8 g +9 codon. A weak toeprint at +24 may indicate a low population of tRNA Gly bound to P site and paired with the g +8 g +9 a +10 glycine codon in the +1 frame. The addition of tRNA Lys (Figure S1A, lane K) triggered translocation leading to a toeprint at +25, which corresponds to a post-translocation complex with tRNA Lys in the P site bound to the a +10 a +11 g +12 lysine codon and leaving an empty A site.