Measuring Programmed Ribosomal Frameshifting

Escherichia coli strain DH5α was used to amplify plasmids, and E.coli transformations were performed using the high efficiency method of Inoue et al. (13 (link)). YPAD and synthetic complete medium (H-) were used as described previously (14 (link)). Yeast strain JD932 (MATa ade2-1 trp1-1 ura3-1 leu2-3,112 his3-11,15 can1-100) (15 (link)) was used for in vivo measurement of programmed −1 ribosomal frameshifting. Yeast cells were transformed using the alkali cation method (16 (link)). Dual luciferase plasmids pJD375 (C₁, no frameshift signal) and pJD376 (F₁, L-A virus gag-pol frameshift signal) have been described previously (7 (link)). Putative frameshift signals from S.cerevisiae genes YOR026W/BUB3 (F₂, plasmid pJD519) and YPL128C/TBF1 (F₃, plasmid pJD478) were constructed as follows: (i) oligonucleotides from Integrated DNA Technology (Coralville, IA) were annealed and gel purified, and (ii) annealing the oligonucleotides 5′-TCGACAAAAAATCATCTTTCAGGGTGGATTGGAACGGCCCCAGTGATCCTGAGAACCCACAAAACTGGCCCG-3′ to 5′-GATCCGGGCCAGTTTTGTGGGTTCTCAGGATCACTGGGGCCGTTCCAATCCACCCTGAAAGATGATTTTTTG-3′ (F₂), and 5′-CGACAAATTTATCTCAAGCATCCTTCATCAGCTGCATCTGCTACTGAAGAGGG-3′ to 5′-GATCCTCTTCTGTAGCAGATGCAGCTGAAGAAGGATGCTGAGATAAATTTG-3′ (F₃) left overhanging single-stranded DNA complementary to SalI and BamHI restriction sites. The annealed oligonucleotides were ligated into p2mci (6 (link)). The frameshift signal was sub-cloned as a SalI–EcoRI fragment into similarly digested pJD375. The open reading frame (ORF) 1a-1b frameshift signal from the SARS-associated Coronavirus (SARS-CoV) was cloned; sense 5′-GATCCTTTTTAAACGGGTTTGCGGTGTAAGTGCAGCCCGTCTTACACCGTGCGGCACAGGCACTAGTACTGATGTCGTCTACAGGGCTTTTGAGCT-3′ and antisense 5′-CAAAAGCCCTGTAGACGACATCAGTACTAGTGCCTGTGCCGCACGGTGTAAGACGGGCTGCACTTACACCGCAAACCCGTTTAAAAAG-3′ oligonucleotides were annealed, gel purified and cloned into BamHI and SacI restricted p2mc (6 (link)). This was further sub-cloned into a pJD375-based plasmid where the reading frame was corrected using site-directed mutagenesis to add a cytosine downstream of the BamHI restriction site (F₄). A zero-frame control (C₂) plasmid was made by inserting two cytosine residues upstream of the BamHI restriction site and cells were grown in the absence or presence of 20 μg/ml of anisomycin (Sigma–Aldrich, St. Louis, MO). The annealed oligonucleotides were ligated into p2mci (6 (link)). The SARS-CoV frameshift signal (F₄) was sub-cloned as a SalI–EcoRI fragment into similarly restricted pJD375. In vivo DLAs for programmed −1 ribosomal frameshifting were performed in yeast strain JD1158 as described previously (7 (link)). Luminescence readings were obtained using a Turner Designs TD20/20 luminometer (Sunnyvale, CA). Reactions were carried out using the Dual-Luciferase® Reporter Assay System from Promega Corporation (Madison, WI).