A Mutation in the gene-encoding bacteriophage T7 DNA polymerase that renders the phage temperature-sensitive

J Biol Chem. 2001 Dec 7;276(49):46151-9. doi: 10.1074/jbc.M106319200. Epub 2001 Sep 10.


Gene 5 of bacteriophage T7 encodes a DNA polymerase essential for phage replication. A single point mutation in gene 5 confers temperature sensitivity for phage growth. The mutation results in an alanine to valine substitution at residue 73 in the exonuclease domain. Upon infection of Escherichia coli by the temperature-sensitive phage at 42 degrees C, there is no detectable T7 DNA synthesis in vivo. DNA polymerase activity in these phage-infected cell extracts is undetectable at assay temperatures of 30 degrees C or 42 degrees C. Upon infection at 30 degrees C, both DNA synthesis in vivo and DNA polymerase activity in cell extracts assayed at 30 degrees C or 42 degrees C approach levels observed using wild-type T7 phage. The amount of soluble gene 5 protein produced at 42 degrees C is comparable to that produced at 30 degrees C, indicating that the temperature-sensitive phenotype is not due to reduced expression, stability, or solubility. Thus the polymerase induced at elevated temperatures by the temperature-sensitive phage is functionally inactive. Consistent with this observation, biochemical properties and heat inactivation profiles of the genetically altered enzyme over-produced at 30 degrees C closely resemble that of wild-type T7 DNA polymerase. It is likely that the polymerase produced at elevated temperatures is a misfolded intermediate in its folding pathway.

Publication types

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

MeSH terms

  • Base Sequence
  • DNA Primers
  • DNA Replication
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / genetics*
  • Escherichia coli / virology
  • Genes, Viral*
  • Hot Temperature*
  • Models, Molecular
  • Mutagenesis
  • Protein Conformation
  • Viral Proteins / biosynthesis


  • DNA Primers
  • Viral Proteins
  • bacteriophage T7 induced DNA polymerase
  • DNA-Directed DNA Polymerase