Interaction of nucleolar phosphoprotein B23 with nucleic acids.
The interaction of eukaryotic nucleolar phosphoprotein B23 with nucleic acids was examined by gel retardation and filter binding assays, by fluorescence techniques, and by circular dichroism. All studies utilized protein prepared under native conditions by a newly developed purification procedure. Electrophoretic gel mobility shift assays with phage M13 DNA suggested that protein B23 is a single-stranded nucleic acid binding protein. This was confirmed in competition binding assays with native or heat-denatured linearized plasmid pUC18 DNA where the protein showed a marked preference for the denatured form. In other competition assays, there was no apparent preference for single-stranded synthetic ribo- versus deoxyribonucleotides. Equilibrium binding with poly(riboethenoadenylic acid) indicated cooperative ligand binding with a protein binding site size of 11 nucleotides and an apparent binding constant (K omega) of 5 x 10(7) M-1 which includes an intrinsic binding constant (K) of 6.3 x 10(4) M-1 and a cooperativity factor (omega) of 800. In circular dichroism (CD) studies, protein B23, when combined with the single-stranded synthetic nucleic acids poly(rA) and poly(rC), effected a decrease in ellipticity and a shift of the positive peak at 260-270 nm toward higher wavelengths, indicating helix destabilizing activity. No CD changes were seen with double-stranded poly(dA.dT). The change in ellipticity of poly(rA) was sigmoidal upon addition of protein, confirming the cooperative behavior seen with fluorescence methods. These studies indicate that protein B23 binds cooperatively with high affinity for single-stranded nucleic acids and exhibits RNA helix destabilizing activity. These features may be related to its role in ribosome assembly.
Dumbar
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Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216-4505.
Gentry
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Olson
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D000818:Animals; D001667:Binding, Competitive; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D008297:Male; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D000090243:Nucleophosmin; D012313:RNA / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013816:Thermodynamics
D004277:DNA, Single-Stranded; D009687:Nuclear Proteins; D000090243:Nucleophosmin; D012313:RNA; D004247:DNA