We subsequently monitored the position of mSP-SL-HIV-1 within the ribosome allowing a single or multiple rounds of EF-G catalyzed translocation. The signal at positions +43 and +47 disappeared to give a new toeprint at +39 (Figure 2 , lane L, and 3B), indicating that the lower stem readily gave way upon just a single translocation step. An identical result was observed in the mSP-SL-HIV-1 RNA which has a wild-type slippery sequence (first codon is u +1 u +2 u +3 ) indicating that codon substitution to a methionine codon did not affect the observed toeprints except for the +47 signal that is ambiguous ( Figure  S1B ). Furthermore upon a single translocation, the +16/+17 toeprint signals disappeared without, however, any occurrence of a new toeprint at position +19 (lane L). The disappearance of the mSP-SL-HIV-1 +16/+17 toeprints upon addition of EF-G is intriguing. We also observed the same phenomenon with the construct containing the wild-type slippery sequence ( Figure S1B ), and interestingly also with an mRNA containing a pseudoknot (BWYV) bound to ribosome (51) . This phenomenon is characteristic to mRNAs containing downstream structural elements, however its cause still need to be elucidated. Ribosomes are known to change conformation upon EF-G binding (52) (53) (54) (55) . One may speculate that this either blocks RT access directly or stabilizes the mRNA structure.