Location of the adenylylation site in T4 RNA ligase

The purification of the enzyme T4 RNA ligase is described from an Escherichia coli strain, KR54, in which the RNA ligase gene (g63) has been inserted into the plasmid pDR540 for inducible expression of g63 from the tac promoter. Adenylylation of the purified enzyme with [14C]rATP followed by digestion with chymotrypsin yielded an adenylylated peptide, the identity of which was determined by fast-atom-bombardment mass spectrometric analysis. The results show that the AMP residue is bound covalently to the lysine at position 99 of the RNA ligase protein sequence.