The P-site tRNA binding triggered a major RT stop at position +17 while only a weak signal at position +16 (the 'classical toeprint'). Previous toeprinting assays under identical conditions but with different phage T4 mRNAs showed a variety of toeprints signals confined to the +14 to +17 range (45) . Thus toeprinting assays are very sensitive to the type of mRNA tested and we note that the toeprint signals with the mSP-HIV-1 and mSP-SL-HIV-1 mRNAs respectively at +16 and +17 fall into this range. Could the observed difference between the two mRNAs be attributed to the hairpin? With unstructured mRNA, we expect a pulling force on the spacer mRNA out of the ribosome purely based on entropic arguments. Such a force presumably exposes base +16 giving rise to the classical toeprint. In the case of the HIV-1 hairpin, physical interactions of the ribosome with the hairpin may provide some sort of strain relieve allowing +16 to slightly relax back into the ribosome. On the other hand, how could such a mechanism be consistent with +16/+17 toeprint obtained when an additional codon was inserted in the spacer, between the AUG codon and the hairpin? If the hairpin provides strain relieve would one not expect the mRNA to recede further back into ribosome, not only protect +16 but also perhaps +17. However, lacking any quantitative information about tension in the spacer, such extra movement may be too small to also protect +17. These are intriguing possibilities and raise questions that require the design of new experiments, well beyond the scope of this work, for answering.