Figure 3

In vitro methyltransferase assays with SETDB1 reveal preferential methylation of Tat lysine 51 and loss binding to cyclin T. A) The panel contains the negative and positive controls for the methylation assay. Both "no substrate" and histone H3 N-terminal mutant (K to A at positions 4, 9, 14, 18, 23, 27, 36, and 37) serve as negative controls. Wild type Tat 1–86 protein was used for in vitro methylation assay. B) The panel shows the incorporation of methyl-³H onto the Tat mutant peptides. Tat K50A showed a ~2 fold drop in counts, whereas the K51A showed more than ~10 fold drop in activity. C) Purified biotin labeled TAR RNA or PolyU RNA was mixed with purified proteins including wild type Tat 1–86, Tat 101, methylated Tat 101, purified Cdk9/cyclin T (data not shown) or extract. Unmodified and methylated Tat (1–86 and 1–101) were incubated with CEM nuclear extract containing endogenous Cdk9/cyclin T complexes (both active and inactive small and large complexes). Biotin-TAR RNA was added to the reaction mixture at the same time, processed and western blotted for presence of cyclin T.