As inferred from the homologous UBA1, activation of ubiquitin by UBA6 (UBE1L1) proceeds through 3 steps: adenylation of ubiquitin, conjugation of ubiquitin from adenyl-ubiquitin to an internal cysteine residue of UBA6, and adenylation of a second molecule of ubiquitin. In the second step, the thiol group of an internal cysteine residue of UBA6 attacks the acyl-adenyl bond of the C-terminal glycine of adenylated ubiquitin, resulting in a thioester bond between the cysteine residue and the C-terminal glycine of ubiquitin (Jin et al. 2007, Pelzer et al. 2007, reviewed in Groettrup et al. 2008).