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Mutational analysis of residues critical to SMYD3 structure and function.

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posted on 14.07.2011, 01:58 by Kenneth W. Foreman, Mark Brown, Frances Park, Spencer Emtage, June Harriss, Chhaya Das, Li Zhu, Andy Crew, Lee Arnold, Salam Shaaban, Philip Tucker

(A) Wild-type SMYD3, but not catalytic point mutant Y239F, methylates recombinant H3 and H4 in an in vitro HMTase assay. Upper panels: Fluorographs with bands corresponding to H3 (left) and H4 (right) indicated. Lower panels: Coomassie-stained PVDF membranes used to verify equal loading. (B) Substitution and (C) truncation mutants, constructed in E coli as described in Methods, were compared in in vitro HMTase assays to wildtype SMYD3 and to SET7/9. Inputs (upper panels, ∼500 ng) were assayed for 3H-SAM incorporation (middle panels) either on recombinant histone 4 or mixed histones, as indicated (lower panels). Alanine substitutions of most SMYD3 residues predicted to be catalytically essential eliminate HMTase activities. An exception is T184/A which, as described in the text, appears to affect H3-H4 substrate specificity (note change in relative ratios of H3/H4). N-terminal truncation through position 44 removes the entire N-SET domain, while truncation through position 74 eliminates both the N-SET domain and half the MYND domain.

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