title: Full-length Dengue virus RNA-dependent RNA polymerase-RNA/DNA complexes: stoichiometries, intrinsic affinities, cooperativities, base, and conformational specificities.
abstract: Fundamental aspects of interactions of the Dengue virus type 3 full-length polymerase with the single-stranded and double-stranded RNA and DNA have been quantitatively addressed. The polymerase exists as a monomer with an elongated shape in solution. In the absence of magnesium, the total site size of the polymerase-ssRNA complex is 26 ± 2 nucleotides. In the presence of Mg(2+), the site size increases to 29 ± 2 nucleotides, indicating that magnesium affects the enzyme global conformation. The enzyme shows a preference for the homopyrimidine ssRNAs. Positive cooperativity in the binding to homopurine ssRNAs indicates that the type of nucleic acid base dramatically affects the enzyme orientation in the complex. Both the intrinsic affinity and the cooperative interactions are accompanied by a net ion release. The polymerase binds the dsDNA with an affinity comparable with the ssRNAs affinity, indicating that the binding site has an open conformation in solution. The lack of detectable dsRNA or dsRNA-DNA hybrid affinities indicates that the entry to the binding site is specific for the sugar-phosphate backbone and/or conformation of the duplex.
authors: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1053, USA.
authors: Michal R
authors: MR
authors: Szymanski
authors: Maria J
authors: MJ
authors: Jezewska
authors: Paul J
authors: PJ
authors: Bujalowski
authors: Cecile
authors: C
authors: Bussetta
authors: Mengyi
authors: M
authors: Ye
authors: Kyung H
authors: KH
authors: Choi
authors: Wlodzimierz
authors: W
authors: Bujalowski
mesh_terms: D004279:DNA, Viral / Q000378:metabolism*; D003716:Dengue Virus / Q000201:enzymology*; D005453:Fluorescence; D007700:Kinetics; D008274:Magnesium; D008958:Models, Molecular; D009692:Nucleic Acid Heteroduplexes; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D012367:RNA, Viral / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000378:metabolism*; D012996:Solutions; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D004279:DNA, Viral; D009692:Nucleic Acid Heteroduplexes; D009711:Nucleotides; D012367:RNA, Viral; D012996:Solutions; D012324:RNA-Dependent RNA Polymerase; D008274:Magnesium