We used a toeprinting assay to monitor the position and/ or structure of the HIV-1 hairpin during the movement of the ribosome along mRNA. This primer-extension inhibition assay has been shown to be a powerful tool for mapping the position of mRNA within 30 S and 70 S ribosomal complexes that contain tRNA (45) . When RT encounters the ribosome (a so-called hard-stop), it terminates cDNA synthesis thereby generating a highly specific toeprint. Alternatively, RT may also stall at mRNA structural elements further downstream from the ribosome that prove too hard for RT to unwind, resulting in what is generally called an extended toeprint.