Extended toeprints (albeit weak ones) at +43 and +47 in the pre-translocation state indicate that a fraction of RTs was incapable of unwinding part of the lower stem and bulge region when encountering the HIV-1 hairpin bound at the ribosome. Since such toeprints do not occur with free hairpins, contacts with the ribosome (34) may be responsible for these signals. Extending the spacer sequence by an extra codon yields an identical toeprint; not surprising as it is hard to imagine that a longer spacer would stand in the way of forming ribosomal contacts. This will be discussed further below. Furthermore, we showed that these toeprints are dependent on the structure of the bulged region of the frameshifting signal. The subsequent replacement of the GGA-bulge with a CCC-bulge, known to reduce -1 PRF efficiency (8, 32) , practically erases the +43 and +47 toeprints, indicating such a mutation affects either contacts with the ribosomes or stability of the hairpin itself. We tend to favor the former possibility as mechanical unfolding of free hairpins indicates the lower stem is fairly weak to begin with. Certainly it remains possible that contact with the ribosome can stabilize part of the lower stem. If such stabilization was to occur, it does not prevent further translocation as a single cycle of EF-G catalyzed translocation moves the toeprint to +39 providing direct evidence for melting of the lower stem. Interestingly, the extended toeprint at +39 remains even under conditions where further translocation is allowed. This suggests that the upper stem is capable of inhibiting translocation of a sizeable fraction of ribosomes. Our mechanical unfolding study supports the notion of a weak lower stem since no transition in the force versus extension curves indicative of unfolding of solely the lower stem have been observed. Although on occasion we suspect such a transition (by visual inspection of the force versus extension data, typically at forces $6 pN and lower), the increase in extension in those cases should be $4-5 nm (by theory), too small to reliably distinguish from thermal fluctuations at those force levels. We note that were these transitions to occur in the steeper part of the force versus extension curve (F > 6 pN), we should be able to detect a shift of the curve toward the right consistent with unfolding of the lower stem. However, the increases in contour lengths ( Figure 5C ) observed are consistent with unfolding of solely the upper stem. Not on any occasions have we observed increases in contour lengths consistent with simultaneous unfolding of the lower and upper stems. Since unfolding is hierarchical, the lower stem has to unfold before the upper stem does. We conclude that the lower stem therefore has much reduced mechanical stability compared to the upper stem. Quantitative assessment of the stability of the lower stem is currently not possible in the existing experimental geometry, and requires higher resolution experimental designs (60) , if at all possible. The question naturally arises how these findings accord with the observed +43 and +47 toeprint and the dependence of frameshifting upon the lower stem and bulge regions. It seems unlikely that tensions as low as a mere 6 pN and below, can trigger a frameshift (61) . Therefore, it is appealing to consider that unfolding of the hairpin in the presence of ribosomes will differ significantly from that when ribosomes are absent. When adhering to a tension-dependent mechanism of -1 frameshifting this suggest that contacts with the ribosome's exterior surface may indeed stabilize the part of the lower stem of the hairpin. Single-molecule mechanical unfolding experiments in the presence of ribosomes are considered, but beyond the scope of this article. Our chemical probing experiments provide further insight into the structure of the HIV-1 frameshift signal when bound to the ribosome. The chemical reactivity pattern indicates that almost the entire structure of the hairpin is maintained. Nucleotides from the ACAA tetraloop and the GGA bulge region remained reactive to chemical probes whereas nucleotides in the upper stem were unreactive. As expected, most of the changes in chemical reactivity of the bases are concentrated in the lower stem. The changes observed in the spacer at positions +7, +8 are consistent with this segment of the RNA being engaged in the 30 S mRNA tunnel (13) . Further downstream in the spacer sequence, nucleotides G +9 , A +10 and G +12 that are expected to be located in the vicinity or at the ribosomal helicase center indeed experienced a change in reactivity. In this segment of the RNA, the DMS reactivity of nucleotide A +11 remained mostly unaffected by the presence of the ribosome. Unfortunately, our chemical probing experiments could not provide useful information on the 3 0 strand of the lower stem. In summary, the data demonstrate that the 5 0 region of the lower stem experiences structural changes when contacted by the ribosome.