Figure 3.

Polypeptide release activity of RF2* at a UAG codon. (A) in vitro fMet release assay with histidine-tagged RF2 and RF2*. f[³H]Met release from the [f[³H]Met-tRNA^f⋅AUG⋅ribosome] complex on addition of RFs and terminator triplets was determined (21). Reactions contained 20 μM UAG (Left), UAA (Center), and UGA (Right), as well as equal molar amounts (50 pmol) of RF proteins. The relative fMet-release activity of RF2* to RF1 at UAG is approximately one-fourth or one-fifth under these experimental conditions (data not shown). (B) The 3A′ reporter gene construct (pAB96) for UAG readthrough assay (15, 16). (C) Influence on UAG readthrough of expression of various RF constructs. Readthrough (RT) values, i.e., molar amounts of 3A′ domain protein (translation readthrough) relative to 2A′ domain protein (translation termination), were measured as described (13, 15, 16). Bars: 1, pSUIQ (vector control); 2, pSUIQT-RF1; 3, pSUIQT-RF2; 4, pSUIQT-RF2*. Experiments were performed independently at least five times in A and C, and the values are expressed with SDs.