Abstract
The RNA ligase RtcB is conserved in all domains of life and is essential for tRNA maturation in archaea and metazoa. Here we show that bacterial and archaeal RtcB catalyze the GTP-dependent ligation of RNA with 3'-phosphate and 5'-hydroxyl termini. Reactions with analogues of RNA and GTP suggest a mechanism in which RtcB heals the 3'-phosphate terminus by forming a 2',3'-cyclic phosphate before joining it to the 5'-hydroxyl group of a second RNA strand. Thus, RtcB can ligate RNA cleaved by RNA endonucleases, which generate 2',3'-cyclic phosphate and then 3'-phosphate termini on one strand, and a 5'-hydroxyl terminus on another strand.