FIGURE 4.

rpr and Dm-hsp70 5′UTR do contain an internal ribosome entry site that functions in vitro. (A) Reporter dicistronic mRNAs used in the in vitro translation assays. rpr-anti indicates a construction in which rpr 5′UTR was cloned in inverse orientation with respect to the second cistron AUG. (RLuc) Renilla luciferase. (B) Normalization of the translation efficiency of dicistronic transcripts. The graph shows the ratio of RLuc activity (translation of the second cistron) to FLuc activity (first cistron) normalized with respect to the same ratio in the FLuc/RLuc transcript. (C) Stability analysis of the dicistronic reporter mRNAs used in B. (D) Reporter dicistronic mRNAs containing the 5′UTR of caudal to prevent readthrough of ribosomes. (E) Translation efficiency of dicistronic transcripts depicted in D is normalized as in B but to the ratio derived from the use of FLuc/cad/RLuc reporter RNA. (F) Stability analysis of the dicistronic reporter mRNAs used in E. (G) Absolute values of translation corresponding to the first cistron (FLuc, white bars) and the second cistron (RLuc, black bars) of the constructs indicated in A and D using m⁷GpppG-capped transcript. (H) Absolute values of translation corresponding to the first cistron (FLuc, white bars) and the second cistron (RLuc, black bars) of the constructs indicated in A and D using ApppG-capped transcript. Note that the scale is one order of magnitude lower than G. Absolute values are comparable, as all of the experiments were performed with the same lot of extracts. The activity of the first cistron decreases dramatically, while the activity of the second cistron remains either constant or increases. (I) Reporter dicistronic mRNAs containing a synthetic, stable hairpin that prevents readthrough of ribosomes. (J) Translation efficiency of dicistronic transcripts depicted in I normalized to the FLuc/cad/RLuc. (K) Stability analysis of the dicistronic reporter mRNAs used in J. Values of all experiments represent at least four independent experiments.