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. Author manuscript; available in PMC: 2019 Dec 7.
Published in final edited form as: J Mol Biol. 2018 Oct 17;430(24):5280–5293. doi: 10.1016/j.jmb.2018.10.003

Fig. 2.

Fig. 2.

RNA enhances TRIM25’s catalytic activity in vitro. (a) Ubiquitination activities of −200 nM TRIM25 purified with (lanes 1, 3, and 6) or without (lanes 2, 4, and 7) PEI precipitation. TRIM25 synthesizes anchored (self-attached) polyubiquitin (polyUb) chains with 1 μM Ube2D3 and unanchored chains with 0.280 μM Ube2N/Ube2V2 [11]. Reactions contained 100 nM E1, 40 μM Ub, and 5 mM Mg-ATP. (b) TRIM25 purified in the absence of PEI treatment was pre-incubated with RNase A (lanes 5 and 9), DNase I (lanes 4 and 8), or buffer control (lanes 3 and 7) prior to setting up ubiquitination assays. (c) TRIM25 purified with PEI treatment was pre-incubated with 500 ng of dsRNA (lanes 4 and 8), 500 ng of dsDNA (lanes 5 and 9), or buffer control (lanes 3 and 7) prior to ubiquitination assays. (d) TRIM25 purified with PEI treatment was pre-incubated with the indicated concentrations of 14, 28, or 56-bp dsRNA prior to ubiquitination with Ube2N/Ube2V2 as E2.