A Mutation in the Gene-encoding Bacteriophage T7 DNA Polymerase That Renders the Phage Temperature-sensitive

Abstract

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 degreesC, 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 degreesC or 42 degreesC. Upon infection at 30 degreesC, both DNA synthesis in vivo and DNA polymerase activity in cell extracts assayed at 30 degreesC or 42 degreesC approach levels observed using wild-type T7 phage. The amount of soluble gene 5 protein produced at 42 degreesC is comparable to that produced at 30 degreesC, 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 degreesC 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.