Wheat RNA ligase contains 5'-hydroxyl kinase, 2',3'-cyclic phosphate 3'-phosphodiesterase, and 5'-phosphate 2'-phosphate-3'-hydroxyl RNA ligase activities in a 110-kDa polypeptide. Taking advantage of a wheat cell-free protein production system, we prepared various fragments containing a part of the enzyme. The method allowed us to check the activities of the fragments rapidly, eliminating the time-consuming cloning and sequencing steps for the expression of the fragment proteins. The results showed that each of the three activities can be assigned to a non-overlapping domain that does not require the presence of the other part(s) of the enzyme for its activity. This contrasts to the case of yeast tRNA ligase, in which the central kinase domain has been suggested to require to be tethered to one of the flanking domains for its activity. (C) 2010 Elsevier Inc. All rights reserved.