Figure 2: Gp5/trx polymerase activity and macromolecular crowding.

(a) T7 DNA polymerase (gp5, yellow) bound to its processivity factor E. coli thioredoxin (trx, red) polymerizes nucleotides continuously on the leading strand. The crystal structure of gp5/trx bound to primer template and an incoming nucleotide (PDB id code: 1t8e (ref. 34) in a view from the side. The figure was created using PyMOL (http://www.pymol.org). (b) Polymerase activity of gp5 with increasing amounts of trx (right). The activity was measured in a standard reaction containing 40 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mM DTT, 50 mM potassium glutamate, 0.25 mM dATP, dCTP, dGTP, and [α–³²P] dTTP, 20 nM primed M13 DNA, 20 nM gp5 and the indicated amount of trx. After incubation at 37 °C for 10 min the amount of [α–³²P] dTMP incorporated into DNA was measured. Increased polymerase activity of gp5 and trx premixed in a ration of 1:1 is observed as the content of PEG is increased (left). (c) Effect of macromolecular crowding by PEG on polymerase activity of gp5/trx in the presence or the absence of salt. Polymerase activity was measured under similar buffer conditions as in a. Gp5 (5 nM) was mixed with trx (25 nM) in the presence of 300 mM NaCl (black) or in absence of NaCl (cyan). The error bars were derived from three independent experiments.