HIV-1 frameshift efficiency is primarily determined by the stability of base pairs positioned at the mRNA entrance channel of the ribosome

Mouzakis, Kathryn D.; Lang, Andrew L.; Vander Meulen, Kirk A.; Easterday, Preston D.; Butcher, Samuel E.

In vitro frameshift assays were completed with each RNA reporter (experimental and positive control) using a Rabbit Reticulocyte Lysate (RRL) System (Promega, nuclease treated, L416A). Differences from our previously described protocol (56) include the following: translation reactions contained 1.25 mg RNA, 10 units of RNasin Plus RNase Inhibitor (Promega, N2615), and 8.75 ml of RRL in 12.5 ml. Following a 90-min incubation at 37 C, reactions were quenched with the addition of 0.5 ml 0.156 M EDTA pH 8.0 (6 mM final), as described previously (28) . For each reporter, a minimum of three independent frameshift assays were completed. Each independent assay included six replicate reactions.