Stem–loop structures can effectively substitute for an RNA pseudoknot in -1 ribosomal frameshifting

Yu, Chien-Hung; Noteborn, Mathieu H.; Pleij, Cornelis W. A.; Olsthoorn, René C. L.

Candidates of interest were constructed in a dual luciferase vector, pDUAL-HIV(0), essentially as described previously (14, 27) . In short, pDUAL-HIV(0) was digested by KpnI and BamHI, followed by insertion of complementary oligonucleotides to clone the SRV-1 gag-pro pseudoknot, various hairpins as shown in Figures 2C and 5, and a negative control (NC) which formed no apparent secondary structure downstream of the slippery sequence. An in-frame control was constructed by inserting an A-residue upstream of the cytosine in the UUUAA AC slippery sequence of a 12 bp hairpin frameshift construct. HeLa cells were cultured in DMEM/high glucose/ stable glutamine (PAA Laboratories GmbH, Germany) and supplemented with 10% fetal calf serum and 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were kept in a humidified atmosphere containing 5% CO 2 at 37 C. Assay protocols were described previously (14) . Briefly, cells were transfected with 300 ng of plasmid using 1 ml of lipofectamine-2000 (Invitrogen) in a 24-well plate. Cells were lysed 24 h after transfection and luciferase activities were quantified by Glomaxmultidetector (Promega, Benelux) according to manufacturer's protocol. Frameshifting efficiency was calculated by dividing the ratio of Renilla luciferase (RL) over Firefly luciferase (FL) activity of the mutant by the RL/ FL ratio of the in-frame control, multiplied by 100.