Figure 2.

(A) Template construct for ribosomal frameshifting. A stop codon is found immediately after the slippery sequence. Because of the requirement for restriction sites, the spacer sequence differs from the wild type. The in vitro template is transcribed by using T7 RNA polymerase at the T7 promoter, and the resulting transcripts are translated by a rabbit reticulocyte lysate in the same reaction tube. For the in vivo ribosomal frameshifting assay, the −1 ribosomal frameshifting elements, including the slippery sequence and the pseudoknot, are inserted between the 5′ β-galactosidase gene and the luciferase gene. A CMV promoter is used in human embryonic kidney cells. The nonframeshifted product is β-galactosidase, whereas frameshifting yields a fusion protein with luciferase. (B) SDS/PAGE analysis of [³⁵S]methionine-labeled translation products from ribosomal frameshifting assay of wild type and selected mutants in the reticulocyte lysate. Translation products are labeled with incoporation of [³⁵S]methionine. The nonframeshifting product (NFS) is the GST protein, and the −1 frameshift product (FS) is a GST–GFP fusion protein.