The covalent attachment of ubiquitin to proteins (ubiquitylation or ubiquitination) plays a fundamental role in the regulation of cellular function through biological events involving cell cycle, differentiation, immune responses, DNA repair, chromatin structure, and apoptosis.

Ubiquitylation is achieved through three enzymatic steps. In an ATP-dependent process, the ubiquitin activating enzyme (E1) catalyzes the formation of a reactive thioester bond with ubiquitin, followed by its subsequent transfer to the active site cysteine of a ubiquitin carrier protein (E2). The selectivity of the ubiquitin cascade for a particular substrate protein relies on the interaction between the E2 conjugating enzyme (of which a cell contains relatively few) and a ubiquitin-protein ligase (E3), of which over 600 have been identified to date.

The E3s are a large, diverse group of proteins, characterized by one of several defining motifs. These include a HECT (homologous to E6-associated protein C-terminus), RING (really interesting new gene) or U-box (a modified RING motif without the full complement of Zn²⁺-binding ligands) domain. Whereas HECT E3s have a direct role in catalysis during ubiquitinylation, RING and U-box E3s facilitate protein ubiquitinylation. These latter two E3 types act as adaptor-like molecules. They bring an E2 and a substrate into sufficiently close proximity to promote the substrate's ubiquitinylation. Although many RING-type E3s, such as MDM2 and c-Cbl, can apparently act alone, others are found as components of much larger multi-protein complexes, such as the anaphase-promoting complex.

Taken together, these multifaceted properties and interactions enable E3s to provide a powerful, and specific, mechanism for protein clearance within all cells of eukaryotic organisms utilising the ubiquitin-proteasome system. The importance of E3s is highlighted by the number of normal cellular processes they regulate, and the number of diseases associated with their loss of function or inappropriate targeting.

E3 ligases also undergo auto-ubiquitinylation, through modification of specific lysine residues within an individual ligase, providing a mechanism thought to be responsible for the regulation of the E3 enzyme itself.