Figure 7.

Binding of 6-phosphogluconate dehydrogenase (6-PGD) to the 3′UTR PAI-1 mRNA. (A) Porcine citrate (CS) and yeast-derived 6-PGD (10 μg) were incubated with the ³²P-labeled PAI-1 mRNA 3′UTR in the presence of tRNA. The reaction mixtures were later digested with RNase T1 and heparin as described in Materials and Methods to avoid nonspecific interaction. After heparin digestion, the reaction mixtures were UV irradiated at 4°C. The immobilized RNA–protein complexes were separated by SDS-PAGE, dried, and autoradiographed. Fp = free probe. (B) Specificity of the yeast 6-PGD–PAI-1 mRNA 3′UTR interaction. Yeast-derived 6-PGD was incubated with a molar excess of unlabeled transcript or homopoly (A), poly (U), poly (G), poly (C) ribonucleotides, or proteinase K or SDS before exposure to ³²P-labeled transcript. The reaction mixtures were later digested with RNase T1 and heparin and subjected to UV cross-linking assay as described in (A).