FIG 4.

Mpe1 binds to RNA with sequence specificity. (A) Coomassie blue-stained gel of purified recombinant Mpe1 proteins used for RNA gel mobility shift assays. (B) RNA gel mobility shift assays were performed with the indicated recombinant proteins as described in Materials and Methods. Concentrations of 300 nM, 600 nM, and 1.8 μM were used for each protein and are indicated by the wedges above the gel. (C) Mapping the binding site of Mpe1 on CYC1 precursor. (Top) Sequence of the CYC1 transcript and positions of signal sequences and the poly(A) site. The locations of sequences complementary to DNA oligonucleotides 1 to 8 are indicated. (Bottom) RNase H protection profile of Mpe1 on ³²P-labeled CYC1 RNA, which was first incubated with binding buffer (no protein [lanes 1 to 9]) or Mpe1 (600 nM [lanes 10 to 18] or 1.8 μM [lanes 19 to 27]) and then cleaved by the addition of RNase H and oligonucleotides as indicated. As a control, oligonucleotides were omitted in lanes 1, 10, and 19. The amounts of digested product were determined for lanes using DNA oligonucleotides 6 to 8 and are indicated under the lane numbers.