Nakaminami, Kentaro , Karlson, Dale, T .
Functional role of the ATP-binding domain in Arabidopsis cold shock domain proteins.
Cold shock domain proteins (CSP) commonly exhibit nucleic acid binding activity. According to the study of CSPs in E. coli and wheat, it is suggested that they function as RNA chaperones during low temperature stress. Under low temperatures, CSPs melt the stabilized secondary structure of RNA and promote translation. However, at the present time the detailed mechanism of nucleic acid melting is not completely known. In the present study, we confirmed that one of the 4 CSPs in Arabidopsis (AtCSP3, At2g17870) contains a consensus p-loop ATP binding motif. The p-loop motif is commonly found in ATP binding proteins. In functional studies investigating DEAD box RNA helicases, it has been shown that nucleic acid binding and DNA unwinding activities occur in an ATP dependent manner. In order to clarify the function of the ATP binding domain of AtCSP3, we initially confirmed whether this protein exhibited ATP binding activity with an ATP pull down assay. In comparison to the other 3 AtCSPs which lack consensus p-loop motifs, AtCSP3 exhibited the highest affinity to the ATP resin. To confirm that the p-loop is the motif that is responsible for ATP binding activity in AtCSP3, we performed site-directed mutagenesis of critical amino acids within the p-loop motif. Investigations with in vitro ATP pull down analysis with recombinant proteins containing mutant p-loop motifs confirmed that the affinity of AtCSP3 to ATP resin is reduced if the p-loop region is mutated. In addition, in vitro nucleic acid binding assays confirmed that mutations of the p-loop inhibited the ability of AtCSP3 to bind ssDNA. When a particular highly conserved lysine residue in the p-loop of AtCSP3 (K68A) was mutated, this resulted in a complete loss of ssDNA binding activity. These data suggest that the ATP binding domain is functionally important for nucleic acid binding activity and may affects its RNA chaperone activity.
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1 - West Virginia University, Division of Plant & Soil Sciences, 1090 Agriculture Science Building, P.O. Box 6108, Morgantown, WV, 26506-6108, USA
2 - West Virginia University, Division of Plant & Soil Sciences
cold shock domain
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM