Single-molecule Taq DNA polymerase dynamics.

Area of Science:

• Biochemistry
• Molecular Biology
• Enzyme Kinetics

Background:

• Taq DNA polymerase is crucial for PCR but its high-temperature dynamics were poorly understood.
• Previous single-molecule techniques were limited in studying fast enzyme motions at elevated temperatures.

Purpose of the Study:

• To investigate the conformational dynamics of Taq DNA polymerase at temperatures up to 85°C.
• To differentiate between whole-enzyme and domain-specific motions during nucleotide incorporation.

Main Methods:

• Utilized single-walled carbon nanotube transistors to monitor Taq polymerase motions in real-time.
• Employed four enzyme orientations to resolve distinct conformational changes.
• Recorded enzyme activity across a temperature range of 22°C to 85°C.

Main Results:

• Observed rapid, transient closures of Taq's fingers domain (20-μs) during nucleotide binding and complementarity testing.
• Distinguished these transient closures from slower, whole-enzyme closures associated with nucleotide incorporation.
• Found minimal temperature dependence for the rate and duration of transient and catalytic events.

Conclusions:

• Taq DNA polymerase exhibits rapid conformational dynamics even at high temperatures.
• The enzyme's temperature sensitivity primarily resides in its rate-determining open state, not in the conformational changes during nucleotide processing.
• This provides new mechanistic insights into Taq polymerase function at physiologically relevant temperatures.