Figure 3.

Edr utilises –1 ribosomal frameshifting to encode two polypeptides. (A) Plasmid templates for in vitro translation. pRF1, pRF2 and pRF1/RF2 expression plasmids were constructed by cloning Edr cDNA fragments into pSP65T as described in Materials and Methods. Plasmids pRF1/RF2-mt1 and pRF1/RF2-mt2 were derived from pRF1/RF2 by introduction of A→G and C→T nucleotide substitutions, respectively, within the putative slippage sequence. pRF1/RF2-mt3 was derived from pRF1/RF2-mt1 and contains an additional deletion of 2 nt predicted to contribute to the stable secondary structure of the pseudoknot structure. Shaded boxes denote the 5′- and 3′-untranslated regions within RF1 and RF2. The heptameric –1 slippage site is denoted in bold. (B) In vitro translation. Separate in vitro transcription and translation of plasmid templates from (A), was performed using rabbit reticulocyte lysate system in the presence of [³⁵S]methionine. Products were subjected to 10% SDS–PAGE prior to autoradiography.