Using circular permutation analysis to redefine the R17 coat protein binding site.
The bacteriophage R17 coat protein binding site consists of an RNA hairpin with a single purine nucleotide bulge in the helical stem. Circular permutation analysis (CPA) was used to examine binding effects caused by a single break in the phosphodiester backbone. This method revealed that breakage of all but one phosphodiester bond within a well-defined 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 isomer with the 5' and 3' ends at the bulged nucleotide bound with wild-type affinity. However, extending the 5' end of this CP isomer greatly reduces binding, making it unlikely that this circularly permuted binding site will be active when embedded in a larger RNA. CPA also locates 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 nucleotides shorter (nucleotides -15 to +2) than the previously determined site (-17 to +2). The smaller binding site was verified by terminal truncation experiments. A minimal-binding fragment (-14 to +2) was synthesized and was found to bind tightly to the coat protein. The site size determined by 3-ethyl-1-nitrosourea-modification interference was larger at the 5' end (-16 to +1), probably due, however, to steric effects of ethylation of phosphate oxygens. Thus, the apparent site size of a protein binding site is dependent upon the method used.
Gott
J M
JM
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309.
Pan
T
T
LeCuyer
K A
KA
Uhlenbeck
O C
OC
D001435:Bacteriophages / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D002213:Capsid / Q000737:chemistry* / Q000378:metabolism; D036022:Capsid Proteins; D005038:Ethylnitrosourea / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry* / Q000378:metabolism*; D000079962:RNA, Circular; D016601:RNA-Binding Proteins
D036022:Capsid Proteins; D000079962:RNA, Circular; D016601:RNA-Binding Proteins; D012313:RNA; D005038:Ethylnitrosourea