When tRNA fMet was bound at the P site, a doublet of toeprints at positions +U16/+G17 was detected ( Figures  S2 and 2A) . We note the existence of a stop at position +17 in the control lane (in the absence of ribosomes). However, the signal in presence of ribosome is substantially stronger and therefore is interpreted as a ribosomal toeprinting signal. tRNA Leu4 was subsequently bound to the A site ( Figures S2, lane 3, and 2A , lane M 0 ) which resulted in the disappearance of the +16 toeprint. The +16 and +17 toeprints are hard stops, independent of the reverse transcription activity indicating that the mRNA secondary structure unfolds during cDNA synthesis as previously seen for a stem-loop (48) and pseudoknots (49) . However, in addition to these hard stops downstream extended toeprints can also exist indicating that mRNA interactions with the ribosome 5'-(N 42 ) aAGGAaauaaa aug uuu aaa cgu aaa ucu (N 68 )-3' can extend beyond the usual 15 nucleotides buried in the ribosome-mRNA track (48, 50) . Unlike hard stops, extended toeprints are generally dependent upon change of temperature, RT concentration, or the source of RT. Therefore, we varied the temperature and also tested different RTs to look for extended toeprints in the case of mSP-SL-HIV-1. Two additional reverse transcription stops were identified within the 3 0 region of the stemloop at positions +47 (very weak) and +43 (Figures 2A  and 3A ). These extended toeprints are weak in comparison to the hard stop at +17 but are absent in the control lane ( Figure 2A , lane C, in the absence of the ribosome). The +43 and +47 toeprints indicate that a fraction of the RT molecules halted when the enzyme approached or encountered bulge region in the HIV-1 hairpin. In order to test the contribution of the bulge region to the extended toeprint, we changed the GGA-bulge by a CCC-bulge (mSP-SL-CCC-HIV-1, Figure 1 ). The intensities of both toeprint signals (at +43 and +47) decreased ( Figure 2B ) and were reproducibly of lower level than the RT stops found in the control lane -70 S. Interestingly, the substitution of the three purines in the bulge by pyrimidines also decreases frameshifting efficiency (8, 32) .