SITE SIZE OF COOPERATIVE SINGLE-STRANDED RNA-BINDING BY POLIOVIRUS RNA-DEPENDENT RNA-POLYMERASE

The poliovirus RNA-dependent RNA polymerase binds cooperatively to sin gle stranded RNA. We have determined the minimal RNA-binding site size of the poliovirus polymerase using binding titration with oligonucleo tides of increasing length. A dramatic increase in affinity was observ ed 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 1 0 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 mo lecules. Direct binding of wildtype polymerase to oligo(U)(12) and oli go(U)(24) RNAs showed differences in affinity and cooperativity consis tent with this model. The increase in binding affinity seen for oligo( U)(10) suggests either that the RNA-binding determinants are widely sp aced on the polymerase structure or that a substantial conformational change in the polymerase occurs upon the filling of its RNA binding si te.