Single-molecule Taq DNA polymerase dynamics
Mackenzie W. Turvey, Kristin Gabriel, Wonbae Lee, Jeffrey Taulbee, Joshua K. Kim, Silu Chen, Calvin J. Lau, Rebecca Elizabeth Kattan, Jenifer T. Pham, Sudipta Majumdar, Davil Garcia, Gregory A. Weiss, Philip G. Collins
Abstract
Taq DNA polymerase functions at elevated temperatures with fast conformational dynamics-regimes previously inaccessible to mechanistic, single-molecule studies. Here, single-walled carbon nanotube transistors recorded the motions of Taq molecules processing matched or mismatched template-deoxynucleotide triphosphate pairs from 22° to 85°C. By using four enzyme orientations, the whole-enzyme closures of nucleotide incorporations were distinguished from more rapid, 20-μs closures of Taq's fingers domain testing complementarity and orientation. On average, one transient closure was observed for every nucleotide binding event; even complementary substrate pairs averaged five transient closures between each catalytic incorporation at 72°C. The rate and duration of the transient closures and the catalytic events had almost no temperature dependence, leaving all of Taq's temperature sensitivity to its rate-determining open state.