Cloning, expression, and characterization of a DNA binding domain of gpNu1, a phage lambda DNA packaging protein

Terminase is an enzyme from bacteriophage lambda that is required for insertion of the viral genome into an empty pro-capsid. This enzyme is composed of the viral proteins gpNu1 (20.4 kDa) and gpA (73.3 kDa) in a holoenzyme complex. Current models for terminase assembly onto DNA suggest that gpNu1 binds to three repeating elements within a region of the lambda genome known as cosB which, in turn, stimulates the assembly of a gpA dimer at the cosN subsite. This prenicking complex is the first of several stable nucleoprotein intermediates required for DNA packaging. We have noted a hydrophobic region within the primary amino acid sequence of the terminase gpNu1 subunit and hypothesized that this region constitutes a protein-protein interaction domain required for cooperative assembly at cosB and that is also responsible for the observed aggregation behavior of the isolated protein. We therefore constructed a mutant of gpNu1 in which this hydrophobic "domain" has been deleted in order to test these hypotheses. The deletion mutant protein, gpNu1DeltaK, is fully soluble and, unlike full-length protein, shows no tendency toward aggregation; However, the protein is a dimer under all experimental conditions examined as determined by gel permeation and sedimentation equilibrium analysis. The truncated protein is folded with evidence of secondary and tertiary structural elements by circular dichroism and NMR spectroscopy. While physical and biological assays demonstrate that gpNu1DeltaK does not interact with the terminase gpA subunit, the deletion mutant binds with specificity to cos-containing DNA. We have thus constructed a deletion mutant of the phage lambda terminase gpNu1 subunit which constitutes a highly soluble DNA binding domain of the protein. We further propose that the hydrophobic amino acids found between Lys100 and Pro141 define a self-association domain that is required for the assembly of stable nucleoprotein packaging complexes and that the C-terminal tail of the protein defines a distinct gpA-binding site that is responsible for terminase holoenzyme formation.     

p53 DNA binding can be modulated by factors that alter the conformational equilibrium

The p53 tumor suppressor protein is a dimer of dimers that binds its consensus DNA sequence (containing two half-sites) as a pair of clamps. We show here that after one wild-type dimer of a tetramer binds to a half-site on the DNA, the other (unbound) dimer can be in either the wild-type or the mutant conformation. An equilibrium state between these two conformations exists and can be modulated by two types of regulators. One type modifies p53 biochemically and determines the intrinsic balance of the equilibrium. The other type of regulator binds directly to one or both dimers in a p53 tetramer, trapping each dimer in one or the other conformation. In the wild-type conformation, the second dimer can bind to the second DNA half-site, resulting in drastically enhanced stability of the p53-DNA complex. Importantly, a genotypically mutant p53 can also be in equilibrium with the wild-type conformation, and when trapped in this conformation can bind DNA.