Since we have constructed seven K-to-R mutant of M[C]Q ubiquitin (i.e.
M[C]Q/K6R, M[C]Q/K11R, and so on), in the future, we will add fluorophore to those proteins to make dye-labeled K-to-R ubiquitin. Then they could be used in ubiquitination assay similar to method Harreman et al. used before [30]. The only difference is we will use fluorescent imaging instead of Western blotting. If we can get the similar result (for example, all mutant except K63R show fluorescent signals of polyubiquitination, and K63R mutant can only form monoubiquitinated product), then this system can be used as a tool to detect and identify ubiquitin chain when investigating ubiquitination system.
Another feasible application of our method is FRET. If Alexa-488 labeled ubiquitin is confirmed to perform monoubiquitination on Pol II, or if K63R mutated ubiquitin is used in our system instead, we can get monoubiquitinated Pol II. Since the monoubiquitinated Pol II can work normally [26], therefore enables us to use it on FRET experiments. A similar method that using FRET to investigate the mechanistic behavior of Pol II has been developed by Chang et al. [42], however, it might be much easier to perform if we can put a donor or acceptor molecule on Pol II just by ubiquitination assay.
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Appendix
Figure A1 Ubiquitination assay in which enzymes failed to catalyze reactions Which reagents were added is listed above. “+” means this reagent was added, “-”
means this reagent was not added. At the row of Ub (ubiquitin), an “F” means
fluorescent-dye-labeled ubiquitin M[C]Q-A488, and a “W” means wild-type ubiquitin.
(A) Silver stain: in this figure, E2-Ub could be observed, indicating E1 had catalytic ability. (B) Western blot. (C) Fluorescent image: in this figure, we can see Ub
conjugated E1 (E1-Ub, red arrow) and E2-Ub (black arrow), but no E3-Ub and Rpb-Ub, indicating the catalytic ability of E2 had lost.
(A) (B)
(C)
57
Figure A2 Ubiquitination assay using Ub D39C-Cy3 (E2, E3 with SUMO tag) Which reagents were added is listed above. “+” means this reagent was added, and “-”
means it was not added. From lane 2 to lane 8, wild-type Ub, wild-type Ub, Ub K63R, Ub D39C-Cy3, Ub D39C/K63R-Cy3, Ub K29R and Ub K48R were used, respectively.
(A) Silver stain
(B) Western blot: polyubiquitination signal could be seen in ubiquitination assay using wild-type Ub, Ub K29R and Ub K48R (arrows).
(C) Fluorescent image: this figure shows that Ub D39C-Cy3 (which has Lys63) could form a smeared band (polyubiquitination) on the top of gel, but Ub D39C/K63R-Cy3 (which does not have Lys63) could only form a single band (monoubiquitination).
(A) (B)
(C)
58
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