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 third step, the UBA1-ubiquitin conjugate adenylates the C-terminal glycine of a second ubiquitin molecule. This results in UBA1 loaded with 2 ubiquitin molecules, one of which is conjugated to an internal cysteine residue of UBA1 and one of which is adenylated and non-covalently bound to UBA1 (Jin et al. 2007, inferred from the rabbit homologue in Haas et al. 1982, Hershko et al. 1983, reviewed in Groettrup et al. 2008).