No abstract

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.

The nucleotide sequence of the gene encoding in the major capsid protein (MCP) of frog virus 3 (FV3) has been determined and compared to other iridovirus capsid genes. Nucleotide sequence and S1 nuclease analysis showed that the FV3 MCP gene encoded a transcript of 1452 nucleotides containing a 12 nucleotide AU-rich 5' nontranslated region (NTR) and a 50-nucleotide 3' NTR whose terminus was predicted to fold into a hairpin of moderate stability. An open reading frame initiating from the the 5'-most AUG codon encoded a protein of 463 amino acids with a predicted molecular weight of 49,860. Expression of the putative FV3 MCP gene in vitro confirmed that it encoded the major capsid protein of FV3 and supported the suggestion that translation initiated at AUG-1. Pairwise amino acid alignments detected a high degree of sequence identity between the FV3 MCP and other iridoviruses. These results indicate that iridoviruses possess an evolutionarily related major capsid protein and provide information useful not only in studies of viral gene expression, but also in characterizing newly isolated iridoviruses.

No abstract