Essential residues in the C terminus of the bacteriophage T7 gene 2.5 single-stranded DNA-binding protein

J Biol Chem. 2006 Sep 1;281(35):25831-40. doi: 10.1074/jbc.M604601200. Epub 2006 Jun 28.


Gene 2.5 of bacteriophage T7 encodes a single-stranded DNA (ssDNA)-binding protein (gp2.5) that is an essential component of the phage replisome. Similar to other prokaryotic ssDNA-binding proteins, gp2.5 has an acidic C terminus that is involved in protein-protein interactions at the replication fork and in modulation of the ssDNA binding properties of the molecule. We have used genetic and biochemical approaches to identify residues critical for the function of the C terminus of gp2.5. The presence of an aromatic residue in the C-terminal position is essential for gp2.5 function. Deletion of the C-terminal residue, phenylalanine, is detrimental to its function, as is the substitution of this residue with non-aromatic amino acids. Placing the C-terminal phenylalanine in the penultimate position also results in loss of function. Moderate shortening of the length of the acidic portion of the C terminus is tolerated when the aromatic nature of the C-terminal residue is preserved. Gradual removal of the acidic C terminus of gp2.5 results in a higher affinity for ssDNA and a decreased ability to interact with T7 DNA polymerase/thioredoxin. The replacement of the charged residues in the C terminus with neutral amino acids abolishes gp2.5 function. Our data show that both the C-terminal aromatic residue and the overall acidic charge of the C terminus of gp2.5 are critical for its function.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Bacteriophage T7 / metabolism
  • DNA, Single-Stranded / metabolism
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • Escherichia coli / metabolism
  • Escherichia coli / virology
  • Gene Deletion
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis
  • Phenylalanine / chemistry
  • Plasmids / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Viral Proteins / chemistry*
  • Viral Proteins / genetics


  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Viral Proteins
  • gene 2.5 protein, Enterobacteria phage T7
  • Phenylalanine