The number of MS2 sites does not determine p53 affinity for RNA. (A) Example RNAs isolated from a yeast three-hybrid screen of a hybrid RNA library containing ~60 nt of random sequence in an expression cassette containing a single MS2 sequence. Hybrid RNAs 1–5/MS2 bind p53 well, while hybrid RNAs 6–9/MS2 do not bind p53. (Left) Growth assay for HIS3 reporter gene expression on selective medium containing a gradient of 0–40 mM 3-AT. (Right) β-Galactosidase specific activities, normalized to the yeast strain expressing RNA 1/(MS2)2. Data reflect at least three independent transformants for each yeast strain. (B) Predicted secondary structures of RNAs that bind p53 (left) and fail to bind p53 (right). MS2 recognition sequences are shown in gray, and unique sequences are highlighted in red. (C) p53 binding does not reflect target RNA abundance. For strains expressing various RNAs (above), yeast total RNA was extracted and assayed by Northern blotting using radiolabeled oligonucleotides complementary to the RNaseP RPR1 leader and to the U6 snRNA as probes. Mobilities of DNA size markers are indicated (left).
