FIGURE 3.

UV cross-linking analysis of proteins binding to MT-1 3′UTR. An [α-³²P] CTP-labeled MT-1 3′UTR RNA probe (50 fmol) was incubated with 35 μg protein (CHO cell S100 extract), prepared as described in Materials and Methods. Complexes were fixed by UV irradiation and the probe was removed by RNase A digestion. Labeled proteins were separated by SDS-PAGE and detected by autoradiography. Lane 1 contains MT-1 3′UTR RNA alone (free probe; FP) and lane 2 shows proteins labeled by the MT-1 3′UTR probe after incubation with S100 extract. Labeled RNA binds to a major band of ∼50 kDa and weaker bands of ∼35–40 and 60–65 kDa. Binding to the protein indicated by the black arrowhead (∼50 kDa) is competed for effectively by a 80–320 molar excess of unlabeled competitor MT-1 3′UTR and Δ76–86 RNAs, but markedly less so by Δ66–76 (lanes 3–11).