title: The helicase-like domain of plant potexvirus replicase participates in formation of RNA 5' cap structure by exhibiting RNA 5'-triphosphatase activity.
abstract: Open reading frame 1 (ORF1) of potexviruses encodes a viral replicase comprising three functional domains: a capping enzyme at the N terminus, a putative helicase in the middle, and a polymerase at the C terminus. To verify the enzymatic activities associated with the putative helicase domain, the corresponding cDNA fragment from bamboo mosaic virus (BaMV) was cloned into vector pET32 and the protein was expressed in Escherichia coli and purified by metal affinity chromatography. An activity assay confirmed that the putative helicase domain has nucleoside triphosphatase activity. We found that it also possesses an RNA 5'-triphosphatase activity that specifically removes the gamma phosphate from the 5' end of RNA. Both enzymatic activities were abolished by the mutation of the nucleoside triphosphate-binding motif (GKS), suggesting that they have a common catalytic site. A typical m(7)GpppG cap structure was formed at the 5' end of the RNA substrate when the substrate was treated sequentially with the putative helicase domain and the N-terminal capping enzyme, indicating that the putative helicase domain is truly involved in the process of cap formation by exhibiting its RNA 5'-triphosphatase activity.
authors.affiliation: Graduate Institute of Agricultural Biotechnology, National Chung Hsing University, Taichung, Taiwan 40227, Republic of China.
authors.forename: Y I
authors.initials: YI
authors.lastname: Li
authors.forename: T W
authors.initials: TW
authors.lastname: Shih
authors.forename: Y H
authors.initials: YH
authors.lastname: Hsu
authors.forename: Y T
authors.initials: YT
authors.lastname: Han
authors.forename: Y L
authors.initials: YL
authors.lastname: Huang
authors.forename: M
authors.initials: M
authors.lastname: Meng
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D017863:Potexvirus / Q000201:enzymology / Q000235:genetics*; D012315:RNA Caps / Q000737:chemistry*; D020365:RNA Helicases / Q000737:chemistry*; D012367:RNA, Viral / Q000737:chemistry*
chemical_list: D012315:RNA Caps; D012367:RNA, Viral; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase; D043583:Nucleoside-Triphosphatase; D020365:RNA Helicases