A plasmid encoding the putative termination-reinitiation signal of MNV (p2luc-MNVwt) was modified by site-directed mutagenesis to include a T3 RNA polymerase promoter 30 bp upstream of the minimal required viral sequence generating plasmid p2luc-MNV-T3. RNA for structure mapping was prepared by in vitro transcription of BamHI-digested p2luc-MNV-T3 using T3 RNA polymerase. Transcription reactions were performed on a 200 ml scale essentially as described [30] . Structure mapping was performed using a 59 endlabelling procedure as described previously [30, 32] . All probing reactions were performed in a final volume of 50 ml and contained ,40,000 c.p.m. 59 33 P-end-labelled transcript, 10 mg Escherichia coli rRNA and the relevant enzymatic or chemical probe. Further details are provided in the legend to Figure 3 . Interaction of the HIV-1 frameshift signal with the ribosome Ribosomal frameshifting on viral RNAs relies on the mechanical properties of structural elements, often pseudoknots and more rarely stem-loops, that are unfolded by the ribosome during translation. In human immunodeficiency virus (HIV)-1 type B a long hairpin containing a three-nucleotide bulge is responsible for efficient frameshifting. This three-nucleotide bulge separates the hairpin in two domains: an unstable lower stem followed by a GC-rich upper stem. Toeprinting and chemical probing assays suggest that a hairpin-like structure is retained when ribosomes, initially bound at the slippery sequence, were allowed multiple EF-G catalyzed translocation cycles. However, while the upper stem remains intact the lower stem readily melts. After the first, and single step of translocation of deacylated tRNA to the 30 S P site, movement of the mRNA stem-loop in the 5′ direction is halted, which is consistent with the notion that the downstream secondary structure resists unfolding. Mechanical stretching of the hairpin using optical tweezers only allows clear identification of unfolding of the upper stem at a force of 12.8 ± 1.0 pN. This suggests that the lower stem is unstable and may indeed readily unfold in the presence of a translocating ribosome. The ribosome translocates mRNA and bound tRNA molecules accurately in order to maintain the reading frame. This process results in movement of the ribosome along the mRNA by three nucleotides toward the mRNA's 3 0 -end. Translocation of mRNA and tRNAs is a property of the ribosome itself (1, 2) , however binding of elongation factor G (EF-G) and subsequent hydrolysis of GTP strongly catalyzes it (3) . Although the ribosome acts as its own helicase, stable folded structures within the coding regions of mRNA affect the rate of translocation, and more seriously, may trigger a change of reading frame (4, 5) . Such frameshifting mRNA elements play a crucial role in the translational control of viral proteins via -1 programmed ribosomal frameshifting (-1 PRF) where the reading frame has shifted by one base toward the mRNA 5 0 -end. The -1 PRF requires both the mRNA slippery sequence at the ribosome coding sites as well as a downstream structural element that resists unfolding, representing a physical barrier to the mRNA translocation machinery. While in many cases the downstream barrier constitutes a hairpin (H)-type pseudoknots (6, 7) , on a rare occasion it can also be a simple stem-loop structure (8) (9) (10) (11) . It is of interest to note that although these pseudoknots or stem-loops also trigger ribosomal pausing at the slippery site, consistent with notion of them acting as physical barriers, the extent of pausing shows no correlation with frameshift efficiency (12) .