THE NUCLEIC-ACID BINDING-ACTIVITY OF NUCLEOLAR PROTEIN B23.1 RESIDES IN ITS CARBOXYL-TERMINAL END

Protein B23 is a major nucleolar phosphoprotein proposed to be a ribos ome assembly factor. Protein B23 exists as two isoforms, B23.1 and B23 .2, differing only in their carboxyl-terminal sequences. The interacti on of recombinantly produced B23 isoforms with double-stranded DNA was studied using gel retardation and nitrocellulose filter disk assays. Protein B23.1 bound saturably to radiolabeled plasmid DNA. By competit ion assays protein B23.1 was also capable of binding RNA and single-st randed DNA. On the other hand, protein B23.2, the shorter of the two i soforms, was not capable of binding double-stranded DNA. The latter re sult suggested that the carboxyl-terminal end of B23.1 is essential fo r DNA binding activity. This was confirmed by partial digestion experi ments using staphylococcal V8 protease which showed that a 5-kDa fragm ent, containing the carboxyl-terminal end of protein B23.1 retained DN A binding activity similar to that of the parent molecule. In contrast , a 19-kDa fragment from the aminoterminal half of B23.1 did not bind DNA. The sequence of the carboxyl-terminal 68 amino acids comprising t he 5-kDa fragment showed little, if any, similarity to other proteins, suggesting that this segment contains a previously undiscovered nucle ic acid binding motif.