The T7 DNA polymerase requires a host factor, E. coli thioredoxin,[1] in order to carry out its function.

As shown in Figure 2, the 3’ hydroxyl group of a primer acts as a nucleophile and attacks the phosphodiester bond of nucleoside 5’-triphosphate (dTMP-PP).

[8] Two Mg2+ ions form an octahedral coordinate network with oxygen ligand and also bring the reactive primer hydroxyl and the nucleotide α-phosphate close together, thereby lowering the entropic cost of nucleophilic addition.

[8] The amino acids present in the active site assist in creating a stabilizing environment for the reaction to proceed.

The Mg2+ ion on the right (Figure 3) interacts with negatively charged oxygens of the alpha(α), beta(β) and gamma(γ) phosphates to align the scissile bond for the primer to attack.

[8] Metal ions and Lys522 contact non-bridging oxygens on the α-phosphate to stabilize the negative charge developing on the α-phosphorus during bond formation with the nucleophile.

Tyr526, His506, Arg518 side chains and the oxygen from the backbone carbonyl group of Ala476 take part in the hydrogen bond network and assist in aligning the substrate for phosphoryl transfer.

In addition to T7 DNA polymerase (also known as gp5), T7 replisome requires only four accessory proteins for proper function: host thioredoxin, gp4, gp2.5, and gp1.7.

Binding of thioredoxin exposes a large number of basic amino acid residues in the thumb region of T7 polymerase.

gp2.5 protects single-stranded DNA produced during replication and coordinates synthesis of leading and lagging strands through interaction between its acidic C-terminal tail and gp5/thioredoxin.

[11] gp1.7 is a nucleoside monophosphate kinase, which catalyzes the conversion of deoxynucleoside 5'-monophosphates to di and triphosphate nucleotides, which accounts for the sensitivity of T7 polymerase to dideoxynucleotides (see Sequenase below).

The stabilization of this region of gp5 allosterically increases the amount of protein surface interaction with the duplex portion of the primer-template.

The resulting thioredoxin-gp5 complex increases the affinity of T7 polymerase for the primer terminus by ~80-fold and acts processively around 800 nucleotide incorporation steps.

During leading strand synthesis thioredoxin-gp5 and gp4 form a high affinity complex increasing overall polymerase processivity to around 5 kb.

However, some studies have provided evidence to suggesting that changes in tension of the template DNA strand caused by base-pair mismatch may induce exonuclease activation.

Wuite et al. observed that applying tension of above 40 pN to the template DNA resulted in 100-fold increase in exonuclease activity.

Cloning with T7 DNA polymerase helps overcome this limitation by allowing digestion of the poly(dT) tract during the second-strand synthesis reaction.