Characterization of the adenylation site in the RNA 3 '-terminal phosphatecyclase from Escherichia coli

RNA 3'-terminal phosphate cyclases are a family of evolutionarily conserved enzymes that catalyze ATP-dependent conversion of the 3'-phosphate to the 2',3'-cyclic phosphodiester at the end of RNA. The precise function of cycl ases is not known, but they may be responsible for generating or regenerati ng cyclic phosphate RNA ends required by eukaryotic and prokaryotic RNA lig ases, Previous work carried out with human and Escherichia coli enzymes dem onstrated that the initial step of the cyclization reaction involves adenyl ation of the protein. The AMP group is then transferred to the 3'-phosphate in RNA,yielding an RNA-N(3')pp(5')A (N is any nucleoside) intermediate, wh ich finally undergoes cyclization. In this work, by using different proteas e digestions and mass spectrometry, we assign the site of adenylation in th e E, coli cyclase to His-309. This histidine is conserved in all members of the class I subfamily of cyclases identified by phylogenetic analysis. Rep lacement of His-309 with asparagine or alanine abrogates both enzyme-adenyl ate formation and cyclization of the 3'-terminal phosphate in a model RNA s ubstrate. The cyclase is the only known protein undergoing adenylation on a histidine residue. Sequences flanking the adenylated histidine in cyclases do not resemble those found in other proteins modified by nucleotidylation .