We next tested an mRNA with an additional codon between the AUG and the start of the lower stem (mSP-Tyr-SL-HIV-1 RNA) extending the spacer, which should at least allow for one round of translocation to be visualized before the ribosome stalls. The toeprints at the new positions A+16/U+17 were unambiguously identified ( Figure S3 ), which places the boundary region contacting the ribosome upstream from the bulge region. These bases are in the upper stem for the mSP-SL-HIV-1 RNA. In Figure 2C , where experimental conditions were tuned as to specifically detect the extended toeprints, the toeprint signals in the region +17 prove somewhat weaker than in Figure S3 , but are always present. As expected, with the longer spacer, a band at +19 appeared upon addition of EF-G ( Figure 2C, lane L) . Interestingly, the extended toeprints did not change and appeared at the exact same positions at A + 46 and U + 50 (corresponding to the A + 43 and U + 47 in mSP-SL-HIV-1 RNA) ( Figure 2C ). Subsequent addition of EF-G and tRNA Tyr and tRNA Gly produced the same changes in the toeprint pattern as for the mSP-SL-HIV-1 RNA (a shift of 4-nt from +46 to +42). We note that in this case the +19 toeprint remains the strongest toeprint in the upper region of the gel (by comparison with the +17 toeprint) ( Figure 2C , lanes Y and G) indicating that translocation, as before, seems to be impaired with the downstream upper stem.