A number of other observations, although of no direct consequence for the interpretation of the extended toeprints, are of interest. First is the observation of the occurrence of an apparent 4-nt translocation step during transfer of tRNA Leu from A site to P site. The disappearance and appearance of respectively the +43 and +39 toeprints indicate that the addition of EF-G triggered a movement of the mSP-SL-HIV-1 RNA inside the ribosome of four nucleotides. However, we note that this 4-nt shift is detected on the 3 0 strand of the mRNA hairpin that is located outside the ribosome and therefore may not necessarily present a 1:1 reflection of what happened at the coding site. On the other hand, single translocation cycles with only the slippery sequence (mSP-HIV-1) also show a 4-nt step. In this experiment we analyzed the toeprint signals that result from A site, P site tRNA binding and subsequent EF-G catalyzed translocation. The mSP-HIV-1 toeprint at +15 corresponds to tRNA Phe that recognizes the overlapping codon, one nucleotide upstream (u -1 u +1 u +2 ). Binding of tRNA Phe to the u -1 u +1 u +2 codon results in an optimal spacing of seven bases between the SD sequence and the start codon (62) . The toeprint at +16 corresponds to tRNA Phe paired to codon 1 (u +1 u +2 u +3 ). Interestingly, overlapping codons u +2 u +3 u +4 and u +3 u +4 u +5 are not pairing with tRNA Phe because no toeprints were observed at +17 and +18. This is most likely due to unfavorable spacing between the P-site codon and the Shine-Dalgarno sequence (47, 63) . After P-site filling and the first round of translocation, the toeprint at +19 is the expected signal for a post-translocation complex with tRNA Leu in the P site bound at the u +4 u +5 a +6 codon. However, the major toeprint signal is at position +20 suggesting that in the post-translocation complex the P site-bound tRNA Leu is interacting with the u +5 a +6 g +7 codon. This corresponds to a 4-nt translocation event. Subsequent translocation cycles seem to force the toeprints back into register. It remains unclear what gives cause to this behavior. We note that a 4-nt translocation has been observed for punctuated mRNA with an extra, unpaired nucleotide between codons (64). Interestingly, Leger and collaborators proposed that slippage and repairing of Psite-bound tRNA Phe in the -1 frame would leave an unpaired nucleotide between the Phe and Leu codons (32) . But this is unlikely at equilibrium in our assay because A-site binding of tRNA Leu mostly repositioned tRNA Phe into the canonical frame removing any unpaired nucleotide between Phe and Leu codons.