Mapping the functional domains of nucleolar protein B23.
Protein B23 is a multifunctional nucleolar protein whose cellular location and characteristics strongly suggest that it is a ribosome assembly factor. The protein has nucleic acid binding, ribonuclease, and molecular chaperone activities. To determine the contributions of unique polypeptide segments enriched in certain classes of amino acid residues to the respective activities, several constructs that produced N- and C-terminal deletion mutant proteins were prepared. The C-terminal quarter of the protein was shown to be necessary and sufficient for nucleic acid binding. Basic and aromatic segments at the N- and C-terminal ends, respectively, of the nucleic acid binding region were required for activity. The molecular chaperone activity was contained in the N-terminal half of the molecule, with important contributions from both nonpolar and acidic regions. The chaperone activity also correlated with the ability of the protein to form oligomers. The central portion of the molecule was required for ribonuclease activity and possibly contains the catalytic site; this region overlapped with the chaperone-containing segment of the molecule. The C-terminal, nucleic acid-binding region enhanced the ribonuclease activity but was not essential for it. These data suggest that the three activities reside in mainly separate but partially overlapping segments of the polypeptide chain.
Hingorani
K
K
Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216, USA.
Szebeni
A
A
Olson
M O
MO
D000818:Animals; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D007700:Kinetics; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000090243:Nucleophosmin; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013816:Thermodynamics
D009687:Nuclear Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D000090243:Nucleophosmin