Abstract
Bacteriophage T4 gene 32 protein, a model for single-strand specific nucleic acid-binding proteins, consists of three structurally and functionally distinct domains. We have studied the effects of the N and C domains on the protein structure and its nucleic acid-interactive properties. Although the presence of the C domain decreases the proteolytic susceptibility of the core (central) domain, quenching of the core tryptophan fluorescence by iodide is unaltered by the presence of the terminal domains. These results suggest that the overall conformation of the core domain remains largely independent of the flanking domains. Removal of the N or the C terminus does not abolish the DNA renaturation activity of the protein. However, intact protein and its three truncated forms differ in DNA helix-destabilizing activity. The C domain alone is responsible for the kinetic barrier to natural DNA helix destabilization seen with intact protein. Intact protein and core domain potentiate the DNA helix-destabilizing activity of truncated protein lacking only the C domain (*I), enhancing the observed hyperchromicity while increasing the melting temperature. Proteolysis experiments suggest that the affinity of core domain for single-stranded DNA is increased in the presence of *I. We propose that *I can "mingle" with intact protein or core domain while bound to single-stranded DNA.
Highlights
Bacteriophage T4 gene 32 protein, first isolated and characterized 30 years ago (1), has been a model for single strandspecific nucleic acid-binding proteins (2). 32 protein1 binds cooperatively to single-stranded nucleic acids, a property essential to its roles in DNA replication, recombination, and repair as well as to its ability to regulate its own expression at the translational level
The Presence of the C-terminal Domain Reduces the Susceptibility to Proteolysis by Endoproteinase Arg-C—We previously showed that the core domain of gene 32 protein was susceptible to the action of mammalian endoproteinase Arg-C, the intact protein was refractory to digestion (7, 9)
We have further explored the effects of the C and N domains on the nucleic acid-interactive properties of gene 32 protein
Summary
32 protein, full-length bacteriophage T4 gene 32 protein; *I, gene 32 protein lacking its C-terminal 48 amino acids; *II, gene 32 protein lacking its N-terminal 21 amino acids; *III, central or “core” domain of 32 protein lacking both C- and N-terminal domains but containing the nucleic acid binding site; LAST motif, (Lys/Arg)3(Ser/Thr) sequence, found twice in 32 protein; poly[d(A-T)], double-stranded polydeoxyribonucleotide with alternating A-T sequence; [DNA]p or [poly[d(A-T)]]p, concentration of DNA in nucleotide residue in mol/liter (M (p)); CT, calf thymus. In 32 protein, it appears that the presence of the C-terminal domain establishes the kinetic barrier to DNA helix destabilization The presence of this domain has a small negative effect on intrinsic nucleic acid binding affinity (3). The quenching of the core domain tryptophan residue fluorescence by iodide is unaltered by the presence or absence of the terminal domains These results further support the notion that the C domain modulates accessibility of large substrates to the nucleic acid binding surface, but the overall conformation of the core domain remains largely independent of the flanking regions. The enhancement of hyperchromicity and increase in Tm is monotonic with increasing amounts of intact protein or *III These results reflect binding of full-length protein or core domain to single-stranded regions created by the action of *I and may be indicative of protein-protein interactions between *I and intact protein or *III
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