Tetrahymena ubiquitin-histone conjugate uH2A. Isolation and structural analysis

The ubiquitin-histone H2A conjugate, uH2A, of the protozoan Tetrahymena pyriformis was isolated by gel chromatography and octadecylsilyl-silica chromatography, from the fractions on the chromatographic purification of histone H2A [Fusauchi, Y. & Iwai, K. (1983) J. Biochem. 93, 1487-1497]. The uH2A showed an amino acid composition corresponding to the sum of an equimolar mixture of two protozoan H2A variants and protozoan free ubiquitin. N- and C-terminal sequencing of the uH2A, by Edman degradation and carboxypeptidase P digestion, showed a branched structure having two N-terminals, those of the H2A and ubiquitin components, and one C-terminal, that of each H2A variant component. Further structural analyses of the uH2A, by tryptic digestion of citraconylated uH2A and of a ubiquitinated BrCN fragment, showed that the ubiquitin C-terminal Gly-Gly is linked to the epsilon-amino group of either Lys-123, 125, or 126 in the H2A sequence, and that the ubiquitin sequence is similar to that of calf thymus but differs at least in the sequence of residues 12-27. The deducted structure was compared with the only known uH2A structure, that of calf thymus, with special reference to the branched site.