Figure 1.

Effect of phosphorylation on CTD binding to DNA and effect of DNA binding on CTD phosphorylation by Cdk7 and Cdk9. (A) DNA binding by different CTD phospho-isoforms. CTD phospho-isoforms were generated by incubating CTD with the respective Cdk in kinase buffer for 2 h at RT. Increasing amounts of CTD and its phospho-isoforms were then incubated with DNA in binding buffers for 30 min at 4°C and then loaded on a 6% native acrylamide gel. Unphosphorylated CTD binds DNA (lanes 2–5). No binding could be detected using CTD phosphorylated by Cdk7 (lanes 6–9) or by both Cdk7 and Cdk9 (lanes 14–17). Binding affinity is strongly decreased when CTD is phosphorylated by Cdk9 (lanes 10–13). (B) CTD and CTD/DNA phosphorylation by Cdk7 and Cdk9. CTD/DNA complex was generated by incubating CTD with a 44 bp double-stranded DNA in binding buffer for 30 min. Kinase assay was performed by incubating 100 ng of each kinase and 5 μg of CTD, either alone or in complex with increasing amount of DNA, in kinase buffer for 5 min at 20°C. CTD phosphorylation by Cdk7 (lane 1) is increased when CTD is in complex with DNA (lanes 2–5). Cdk9 prefers CTD (lane 6) over CTD/DNA (lanes 7–10) as substrate. Cdk2 phosphorylation by Cdk7 is not affected by DNA (lanes 11–15). Once phosphorylated, Cdk2 is able to phosphorylate Cdk7 (faint band just above Cdk2 band). Cdk7 is produced in baculo-viral-infected insect cells and reproducibly purified as 70% monophosphorylated and 30% bisphosphorylated (18).