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. 2005 Mar 14;33(5):1553–1563. doi: 10.1093/nar/gki299

Figure 1.

Figure 1

Construction of plasmid pKT0/Edr. A 1230 bp DNA fragment (631–1861) encompassing the Edr frameshift region was amplified by PCR from plasmid pSP64T/Edr (20) and cloned into NcoI/HindIII digested plasmid pKT0 (28). The 5′ and 3′ portions of the cloned Edr segment are shown in lower case, numbered according to the mRNA sequence (A of natural AUG start site is base 452; accession no. AJ006464). The AUG for the expression of Edr sequences in pKT0/Edr is derived from the vector (upper case, underlined). In ribosomal frameshifting assays, capped mRNAs were prepared by SP6 transcription of NdeI-linearized templates (unless otherwise stated). The predicted size of the non-frameshifted (stop) and frameshifted (fs) products generated from the translation of this mRNA in RRL is 29 and 32 kDa, respectively. For structural analysis of the Edr stimulatory RNA, a T3 promoter was introduced into the Edr sequence (at position 1357) to generate plasmid pKT0/Edr/T3. Linearization of this plasmid with NdeI and subsequent transcription with T3 RNA polymerase yields a transcript of 152 nt.