In vitro dissection revealed that the kinase domain of wheat RNA ligase is physically isolatable from the flanking domains as a non-overlapping domain enzyme |
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Authors: | Shin-ichi Makino Tatsuya Sawasaki Yaeta Endo Kazuyuki Takai |
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Affiliation: | a Cell-free Science and Technology Research Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan b Venture Business Laboratory, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan c Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan |
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Abstract: | 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. |
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Keywords: | Multidomain enzyme Wheat RNA ligase Domain structure Cell-free translation |
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