Ubiquitination of an artificial RING finger without a substrate and a tag.

Alpha-helical region substitution was applied to the SIAH1 and EL5 RING fingers. The Williams-Beuren syndrome transcription factor (WSTF) PHD_SIAH1 and WSTF PHD_EL5 RING fingers were created as the artificial ubiquitin-ligating enzyme (E3). These fingers possess E3 activities of mono-ubiquitination and poly-ubiquitination, respectively, with ubiquitin-conjugating enzyme (E2)-binding capabilities. Artificial E3s bind two zinc atoms and adopt a zinc-dependent ordered structure and ubiquitinate upon themselves without a substrate and a tag. Ubiquitination experiments using biotinylated ubiquitin showed that the WSTF PHD_EL5 RING finger is poly-ubiquitinated via residue Lys(63) of ubiquitin. Substitution of alpha-helical region might be applicable to various RING fingers with mono-ubiquitination or poly-ubiquitination.