Fig 4.

KlEst1 interacts directly with the putative Est1 binding site of telomerase RNA. (A) Est1_NTD was expressed in E. coli and purified as described in Materials and Methods. The purified protein was analyzed by SDS-PAGE, followed by Coomassie staining (left) or Western blotting using antibodies against the FLAG tag fused to the C terminus of the recombinant protein (right). (B) The secondary structure of Ter1_EBD as predicted by RNAfold is displayed. The mutations tested as shown in panel C, including B-del, SL-del, B-sub, and CS2a-sub, are indicated. (C) Purified Est1_NTD (12.5 nM) was incubated with ³²P-labeled Ter1_EBD, antisense Ter1_EBD (α-Ter1_EBD), or mutants of Ter1_EBD (16 nM) and subjected to UV irradiation and RNase digestion. The products were analyzed by SDS-PAGE and PhosphorImager scanning. Representative assays are shown at the top and the summary data from three independent experiments are shown at the bottom.