FIGURE 4.

Effects of the temperature-sensitive mutant allele of eRF1 (Sup45 I222S) on PRF in yeast cells. A, yeast strain and strategy used for analyzing the effect of eRF1 mutation on the HIV-1 PRF. Yeast cells, from which endogenous eRF1 was deleted, were transformed with a plasmid harboring either wild type eRF1 or temperature-sensitive eRF1 alleles and various dual luciferase constructs. After a 1-h incubation at 37 °C to deactivate temperature-sensitive eRF1, fluc and rluc activities were determined and relative frameshifting ratios obtained. B, reporter constructs. Basic backbone of these constructs was made by inserting the HIV-1 PRF between the two luciferase genes with all of the constructs using the PGK1 promoter in the yeast pRS416 plasmid. Slip represents the reporter construct with fluc in the −1 reading frame relative to rluc. PC is a positive control vector in which rluc and fluc are in the same reading frame; NC represents a negative control in which a stop codon was inserted between rluc and fluc 5′ to the PRF signal. C, relative frameshifting ratio determined by dividing fluc activity by the rluc activity normalizing the samples to wild type eRF1 plus slip construct sample. Each value was average from three independent samples. WT, wild type; MT, mutant. Error bars, ±S.D.