Proteins catalyzing the final step of tRNA splicing-ligation of tRNA exon halves [35]-have first been identified by biochemical fractionation approaches in yeast [50, 51] and plants [52] . Initial biochemical experiments already indicated that the ligation step is mechanistically not as conserved among the different archaeal and eukaryal organisms as the endonuclease reaction [53] [54] [55] [56] . Interestingly, although not requiring tRNA splicing enzymes, Escherichia coli has been shown to catalyze ligation of tRNA exon halves [57] . Even earlier, an RNA ligase activity had been identified in bacteriophage T4-infected E. coli [58] . These experiments led to the conclusion that, based on the type of phosphodiester bond established, distinct types of RNA ligase activity exist [10] (Fig. 2B ). Bacteriophages on one hand, and fungi and plants on the other, utilize two related but mechanistically distinct multistep reactions to prepare the 2 0 ,3 0 -cyclic phosphate and 5 0 -hydroxyl termini produced by the upstream endonuclease reaction for ligation (Fig. 2B , left box). Both mechanisms require the hydrolysis of the 2 0 ,3 0 -cyclic phosphate and phosphorylation of the 5 0 -hydroxyl prior to ligation and as a result the phosphate at the newly formed phosphodiester linkage originates from the nucleotide triphosphate cofactor used for the kinase reaction [10] . To indicate that a 5 0 -phosphate is joined to a 3 0 -hydroxyl Diversity and roles of (t)RNA ligases 2659 moiety, these two pathways are hereinafter referred to as 5 0 -3 0 RNA ligase mechanisms. In addition, two mechanisms exist that incorporate the cleavage site-derived phosphate into the splice junction (Fig. 2B , central and right boxes). 3 0 -5 0 RNA ligation converts 2 0 ,3 0 -cyclic phosphate and 5 0 -hydroxyl termini into a 3 0 ,5 0 phosphodiester and can Appp adenosine 5 0 -triphosphate; App adenosine 5 0 -diphosphate; Ap adenosine 5 0 -monophosphate; Gppp guanosine 5 0 -triphosphate; Gpp guanosine 5 0 -diphosphate; Nppp unspecified nucleoside 5 0 -triphosphate; Np unspecified nucleoside 5 0 -monophosphate; pp pyrophosphate; Ap-Lig adenylated ligase protein; NT-domain nucleotidyl transferase domain; Ptase 2 0 -phosphotransferase; NAD ? nicotinamide adenine dinucleotide; Appr[p ADP-ribose-1 00 ,2 00 -cyclic phosphate be detected in archaeal and vertebrate cell extracts. During 2 0 -5 0 RNA ligation, predominantly found in eubacteria and archaea, 2 0 ,3 0 -cyclic phosphate and 5 0 -hydroxyl moieties are joined to give rise to a 2 0 ,5 0 -phosphodiester bond [59, 60] . 5 0 -3 0 RNA ligases in bacteriophages, bacteria, archaea, and kinetoplastids