Activation of ubiquitin by UBA1 proceeds through 3 steps: adenylation of ubiquitin, conjugation of ubiquitin from adenyl-ubiquitin to an internal cysteine residue of UBA1, and adenylation of a second molecule of ubiquitin. In the second step, the thiol group of an internal cysteine residue of UBA1 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, inferred from the rabbit homologue in Haas et al. 1982, Hershko et al. 1983, reviewed in Groettrup et al. 2008).