Effects of Point Mutation and Deletion of the Capping Enzyme of Bamboo Mosaic Virus on its Methyltransferase and AdoMet-dependent Guanylyltransferase Activities
Autor: | Kai-Pin Tang, 唐愷嬪 |
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Rok vydání: | 2007 |
Druh dokumentu: | 學位論文 ; thesis |
Popis: | 95 Bamboo mosaic virus is a positive-strand RNA virus. Open reading frame 1 of this virus encodes a ~155 kDa replicase that contains a capping enzyme domain, a helicase-like domain, and a RNA dependent RNA polymerase. The capping enzyme posses activity of AdoMet-dependent Guanylyltransferase (GTase) which consists of GTP methyltransferase(MTase) and m7GTP:RNA Guanylyltransferase. GTP methyltransferase can form the m7GTP by transferring a methyl group from AdoMet to the N7 position of GTP, followed by the guanylyltransferase activity to transfer the m7GMP moiety of the m7GTP to the 5’-diphosphate terminus of the viral RNA. In this study, The MTase activities of H66S and H66T are 12% and 5% of that of WT respectively; and the GTase activities are 0.6% and ~0% of that of WT, respectively. The MTase and GTase activity of K121R is similar to those of WT respectively. D122E and D122N have 10% and 7% MTase activity of WT respectively; but lose GTase activity both. R125K has 11% MTase activity of WT, but loses GTase activity. Y213F and Y213M have 26% and 4% MTase activities of WT respectively; and the GTase activities are 0.18% and 0.1% of that of WT, respectively. In order to increase the activity of capping enzyme and define the domain boundary, I deleted terminus region, 1~11, 1~18, 1~20, 423~442 and 434~442. The 434~442 region truncated capping enzyme has ~1.7 fold the GTase activity of WT. However, the GTase activity of the other truncated capping enzymes can’t be detected. Previous result of peptide mapping suggested that N283 might covalently bond to the m7GMP. Nevertheless, the GTase activity of the mutant protein, N283A, is similar to that of WT, indicating that N283 is not the residues that bind the m7GMP covalently. |
Databáze: | Networked Digital Library of Theses & Dissertations |
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