Bacteriophage T7 deoxyribonucleic acid replication invitro. Bacteriophage T7 DNA polymerase: an an emzyme composed of phage- and host-specific subunits.

Abstract:

The DNA polymerase induced after infection of Escherichia coli by phage T7 has been purified 500-fold to near homogeneity as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme complements extracts of cells infected with a T7 gene 5 mutant to permit cell-free replication of duplex T7 DNA. In contrast, purified T4 DNA polymerase or E. coli DNA polymerase I is unable to do so, thus suggesting a specific requirement for the T7 enzyme in the replication of the viral DNA. E. coli TsnC protein is present in purified T7 DNA polymerase in one-to-one stoichiometry with T7 gene 5 protein, and can be isolated in homogeneous form from heat-denatured enzyme by chromatography on DEAE-cellulose. The inactive form of T7 gene 5 protein that accumulates in tsnC hosts has been partially purified. When partially purified gene 5 protein is mixed with purified TsnC protein, DNA polymerase activity is restored, and formation of a one-to-one complex between the two proteins occurs. These results indicate that the functional form ofT7 DNA polymerase is a complex composed of phage- and host-specified subunits.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...)

One line summary:

This paper shows that T7 DNA polymerase functions when T7 gene 5 protein and E. coli TsnC (thioredoxin) are combined and that both subunits are required for replication

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T7 Modrich P1975 Molecular Weight 9.6E+04 Dalton Technique: SDS-PAGE (2 subunits: 84 kD gene 5 protein + 12 kD TsnC protein)
T7 Modrich P1975 Cloned or native Native organism
T7 Modrich P1975 Tagged No
T7 Modrich P1975 Full length or truncated Full length
T7 Modrich P1975 Specific Activity 1750 units/mg

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