USING CIRCULAR PERMUTATION ANALYSIS TO REDEFINE THE R17 COAT PROTEIN-BINDING SITE

The bacteriophage R17 coat protein binding site consists of an RNA hai rpin with a single purine nucleotide bulge in the helical stem. Circul ar permutation analysis (CPA) was used to examine binding effects caus ed by a single break in the phosphodiester backbone. This method revea led that breakage of all but one phosphodiester bond within a well-def ined binding site substantially reduced the binding affinity. This is probably due to destabilization of the hairpin structure upon breaking the ribose phosphates at these positions. One circularly permuted iso mer with the 5' and 3' ends at the bulged nucleotide bound with wild-t ype affinity. However, extending the 5' end of this CP isomer greatly reduces binding, making it unlikely that this circularly permuted bind ing site will be active when embedded in a larger RNA. CPA also locate s the 5' and 3' boundaries of protein binding sites on the RNA. The 5' boundary of the R17 coat protein site as defined by CPA was two nucle otides shorter (nucleotides -15 to +2) than the previously determined site (-17 to +2). The smaller binding site was verified by terminal tr uncation experiments. A minimal-binding fragment (-14 to +2) was synth esized and was found to bind tightly to the coat protein. The site siz e determined by 3-ethyl-1-nitrosourea-modification interference was la rger at the 5' end (-16 to +1), probably due, however, to steric effec ts of ethylation of phosphate oxygens. Thus, the apparent site size of a protein binding site is dependent upon the method used.