Site size of cooperative single-stranded RNA binding by poliovirus RNA-dependent RNA polymerase.
The poliovirus RNA-dependent RNA polymerase binds cooperatively to single-stranded RNA. We have determined the minimal RNA-binding site size of the poliovirus polymerase using binding titration with oligonucleotides of increasing length. A dramatic increase in affinity was observed when the length of the oligo(U) increased from 8 to 10 nucleotides (nt), arguing that the minimal size of RNA for polymerase binding is 10 nt. Another increase in affinity seen as the oligo(U) reached 24 nt suggests that a 24-nucleotide RNA can be occupied by two polymerase molecules. Direct binding of wild-type polymerase to oligo(U)12 and oligo(U)24 RNAs showed differences in affinity and cooperativity consistent with this model. The increase in binding affinity seen for oligo(U)10 suggests either that the RNA-binding determinants are widely spaced on the polymerase structure or that a substantial conformational change in the polymerase occurs upon the filling of its RNA-binding site.
Beckman
M T
MT
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309, USA.
Kirkegaard
K
K
D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D017955:Poliovirus / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase; D014764:Viral Proteins / Q000378:metabolism
D004268:DNA-Binding Proteins; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D012313:RNA; C090443:polymerase 3Dpol, poliovirus; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases