Figure 4.

Nuclease activity assays for purified Dom34 (and its domains). (A) Coomassie-stained protein gel of purified Dom34 and Hbs1. (B) Coomassie-stained protein gel of purified Dom 34 domains (MBP-domain 1 and MBP-domain 2 conjugates). (C) RNA cleavage assay with various expressed proteins. Bulk RNA (from yeast) was incubated with 1 μM of the indicated protein for 1 h at 40°C. Reactions were analyzed on a 1.2% formaldehyde-agarose gel. (D) Analysis of cleavage products from 3′ end-labeled hairpin RNA. Radiolabeled RNA containing a stable 34-base pair hairpin was incubated (alone, with MBP-domain 1 or with MBP-domain 2) for indicated times and cleavage products were analyzed on a 7 M urea/6% polyacrylamide gel.