Fig 2.
Impact of spacer sequences downstream of uORF2 on initiation events at the AUGELK-1. (A) Schematic representation of the ΔKpn-FLuc/β-actin–RLuc reporters. The NheI indicates a unique restriction site introduced just downstream of the uORF2. The spacer elements of 13, 26, and 50 nt were inserted at this site. The 14 nt is the normal distance between the uORF2 and AUGELK-1. (B) Constructs were cotransfected into HEK293T cells with the β-actin–RLuc control. Normalization of reporter activities was performed as outlined in Materials and Methods. For the 5′ UTR spacer series, the SPACER 0 (no insert) value was set at 100. (C) Reinitiation frequency was estimated for SPACER 0 and SPACER 50 by comparing the normalized FLuc/RLuc ratios for the WT and UGA/C constructs, as outlined in the legend to Fig. 1. The mean percent reinitiation frequency is indicated below each column. All transfections were performed in triplicate, and the bars indicate the SEM. In panel C, P = 0.001.