MUTATIONAL ANALYSIS OF THE PROHEAD BINDING DOMAIN OF THE LARGE SUBUNIT OF TERMINASE, THE BACTERIOPHAGE LAMBDA DNA PACKAGING ENZYME

摘要

Terminase, the DNA packaging enzyme of bacteriophage lambda, is made up of two subunits, gpNul and gpA, the products of the Nu1 and A genes. The activities of terminase include DNA binding, cos cleavage and prohead binding. Specificity domains within the structure of terminase have previously been defined by genetic studies of lambda-21 hybrids. The prohead binding domain of terminase is localized to the last 32 amino acid residues of gpA. Mutations in the prohead binding domain of gpA were constructed by introducing the corresponding amino acids from gp2, the gpA analog of bacteriophage 21. The last five residues of gpA can be replaced with little effect on the burst size of lambda. A phage with a replacement of the last six residues of gpA with the corresponding residues of gp2 was unable to form plaques, indicating that the sixth-to-last residue of gpA is crucial for prohead binding. Site-specific mutagenesis of the sixth-to-last position of gpA indicated that the sixth-to-last residue of gpA must be hydrophobic, of the seven amino acids tested, only isoleucine and valine can substitute for leucine at this position Although the last five residues of gp2 were functional when they replaced the last five residues of gpA, two results indicated that the last five residues of gpA functioned better than the corresponding residues of gp2. First, the presence of a valine residue at the sixth-to-last position of gpA allowed plaque formation, whereas replacement of the last six residues of gpA with those of gp2, which substitutes a valine residue at the sixth-to-last position, was lethal. The second set of results indicating that the last five residues of gpA function better than the gp2 residues were obtained by study of revertants of lethal substitution mutations. In constructing the replacement mutations, a short linker was inserted into the C terminus of the A gene; this insertion created a short duplication of the end of the A gene, so that the normal C-terminal codons were located downstream of the stop codon of the A gene in the substitution mutants. Revertants of the lethal subsitution mutations were obtained in which a mutation in the stop codon resulted in addition of the last five residues of gpA to the end of the substitution terminase. Thus lethal mutants with six or seven residues of gp2 substituted for the normal C terminus of gpA were restored to viability by addition of the normal five residues at the C terminus of the mutant gpA. Such revertants also indicate positional flexibility for the terminal residues of the prohead binding domain: the last five residues of gpA can suppress a lethal substitution when added to the end of the polypeptide. [References: 49]