A Mutation in the gene-encoding bacteriophage T7 DNA polymerase that renders the phage temperature-sensitive.
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.
Kumar
J K
JK
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Kremsdorf
R
R
Tabor
S
S
Richardson
C C
CC
D001483:Base Sequence; D017931:DNA Primers; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000821:virology; D005814:Genes, Viral; D006358:Hot Temperature; D008958:Models, Molecular; D016296:Mutagenesis; D011487:Protein Conformation; D014764:Viral Proteins / Q000096:biosynthesis
D017931:DNA Primers; D014764:Viral Proteins; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase