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. 2016 Dec 6;45(3):1371–1391. doi: 10.1093/nar/gkw1234

Figure 3.

Figure 3.

Activity of SmYbeY on dsRNA. Enzymes were used at a concentration of 5 μM in all assays. Reactions were incubated in KCl-containing buffer in the presence of the co-factor indicated on top of the panels. Reaction times are also indicated. Cleavage products were separated on 7 M urea/20% polyacrylamide gels. C, control reactions. (A) In vitro reactivity of purified wild-type SmYbeY and the mutant variant SmYbeY-R69A on a partially dsRNA substrate (30:16-ds) (0.02 pmol/μl) whose sequence is indicated on bottom. The bracket to the left indicates the size range of breakdown products. RNase E was assayed with the 30:16-ds and 30mer ssRNA substrates (0.02 pmol/μl) as control of cleavage specificity on dsRNA (right panel). The gel of a shift assay (15% polyacrylamide) showing effective duplex formation is shown on bottom. (B) Reactivity of SmYbeY and SmYbeY-R69A on the 39:39-ds dsRNA substrate (0.24 pmol/μl), whose sequence is shown on bottom. Gel of the shift assay (15% polyacrylamide) showing formation of the duplex is also shown.