RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. A RING finger-containing protein, AO7, was identified that binds ubiquitin-conjugating enzymes (E2s) and is a substrate for E2-dependent ubiquitination. Mutations of cation-coordinating residues within AO7's RING finger abolished ubiquitination, as did chelation of zinc. Several RING finger proteins, including BRCA1, Siah-1, TRC8, NF-X1, kf-1, and Praja1, were assessed for their ability to facilitate E2-dependent ubiquitination. In all cases, ubiquitination was observed. The RING fingers were implicated directly in this activity through mutations of metal-coordinating residues or chelation of zinc. These findings suggest that a large number of RING finger-containing proteins may play previously unappreciated roles in modulating protein levels via ubiquitination. Ubiquitination is a primary mechanism by which proteins are targeted for proteasomal degradation. It involves the sequential action of Ub-activating enzyme (E1), Ub-conjugating enzymes (E2s), and Ub protein ligases (E3s). E3s recognize E2 and facilitate the transfer of Ub from E2 to substrate. E3s play important roles in catalyzing the formation of chains of Ub molecules on substrates, which are crucial for recognition by proteasomes. Despite the large number of substrates, relatively few E3s have been characterized on a molecular level. E3s for which the amino acid sequences are known include the N end rule E3s of yeast and mammals, and members of the HECT family. Mammalian HECT E3s include E6-AP and Nedd4. In an attempt to identify additional Ub protein ligases, a member of a family of highly conserved human core E2s was used in a yeast two-hybrid screen. This screen resulted in the identification of a previously uncharacterized RING finger protein, AO7, that undergoes ubiquitination in the absence of eukaryotic proteins other than E1 and E2. Characterization of AO7 led to the determination that six additional, otherwise-unrelated RING proteins also have the capacity for E2-dependent ubiquitination. AO7 undergoes E2-dependent ubiquitination. To determine whether AO7 might have a role in ubiquitination, an in vitro assay was used. This assay employs recombinant proteins expressed in E. coli that do not express components of the Ub-conjugating system. Proteins being evaluated were expressed as GST fusions and isolated by binding to GS. 32P-labeled recombinant Ub and recombinant E1 and E2 were added. GST fusion proteins serve as potential substrates. Evaluation of GST-AO7 revealed substantial ubiquitRING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. A RING finger-containing protein, AO7, was identified that binds ubiquitin-conjugating enzymes (E2s) and is a substrate for E2-dependent ubiquitination. Mutations of cation-coordinating residues within AO7's RING finger abolished ubiquitination, as did chelation of zinc. Several RING finger proteins, including BRCA1, Siah-1, TRC8, NF-X1, kf-1, and Praja1, were assessed for their ability to facilitate E2-dependent ubiquitination. In all cases, ubiquitination was observed. The RING fingers were implicated directly in this activity through mutations of metal-coordinating residues or chelation of zinc. These findings suggest that a large number of RING finger-containing proteins may play previously unappreciated roles in modulating protein levels via ubiquitination. Ubiquitination is a primary mechanism by which proteins are targeted for proteasomal degradation. It involves the sequential action of Ub-activating enzyme (E1), Ub-conjugating enzymes (E2s), and Ub protein ligases (E3s). E3s recognize E2 and facilitate the transfer of Ub from E2 to substrate. E3s play important roles in catalyzing the formation of chains of Ub molecules on substrates, which are crucial for recognition by proteasomes. Despite the large number of substrates, relatively few E3s have been characterized on a molecular level. E3s for which the amino acid sequences are known include the N end rule E3s of yeast and mammals, and members of the HECT family. Mammalian HECT E3s include E6-AP and Nedd4. In an attempt to identify additional Ub protein ligases, a member of a family of highly conserved human core E2s was used in a yeast two-hybrid screen. This screen resulted in the identification of a previously uncharacterized RING finger protein, AO7, that undergoes ubiquitination in the absence of eukaryotic proteins other than E1 and E2. Characterization of AO7 led to the determination that six additional, otherwise-unrelated RING proteins also have the capacity for E2-dependent ubiquitination. AO7 undergoes E2-dependent ubiquitination. To determine whether AO7 might have a role in ubiquitination, an in vitro assay was used. This assay employs recombinant proteins expressed in E. coli that do not express components of the Ub-conjugating system. Proteins being evaluated were expressed as GST fusions and isolated by binding to GS. 32P-labeled recombinant Ub and recombinant E1 and E2 were added. GST fusion proteins serve as potential substrates. Evaluation of GST-AO7 revealed substantial ubiquit