Selection of hybrid RNAs that bind strongly to p53 in the yeast three-hybrid system. (A) Yeast three-hybrid screen of a hybrid RNA library containing ~60 nt of random sequence yielded four clones [hybrid RNAs 1–4/(MS2)2] with improved p53 binding compared to –/(MS2)2 or IRE/(MS2)2. (Left) Growth assay for HIS3 reporter gene expression on selective medium containing a gradient of 0–40 mM 3-AT. (Right) RNAs –/(MS2)2, IRE/(MS2)2, and 1– 4/(MS2)2 do not activate reporter genes in the absence of p53. (B) Sequences of the selected random inserts (shown 5′ to 3′) within hybrid RNAs 1–4. Blocks of nucleotide identity between aligned sequences (1 and 3) and (2 and 4) are highlighted in black. (C) Predicted secondary structures of hybrid RNAs 1–4. MS2 recognition sequences are shown in gray. Unique sequences in each RNA are highlighted in red. (D) LacZ activation is independent of RNA length. Data points reflect the mean of at least three independent transformants for each yeast strain.
