We then applied this assay to a gene 32 message (mSP-SL-HIV-1), which contains the mRNA frameshifting signal (Figure 2A) . In order to avoid any unnecessary ambiguity in interpreting the toeprints, we substituted the phenylalanine codon (u +1 u +2 u +3 ) for a methionine codon (a +1 u +2 g +3 ) (mSP-SL-HIV-1; Figure 1 ) to avoid complications due to the presence of the slippery site. This allows tRNA binding in only a single unique reading frame.