T7 RNA polymerase studied by force measurements varying cofactor concentration

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• RNA polymerases synthesize RNA from a DNA template, translocating along the DNA and incorporating nucleotide triphosphates (NTPs).
• Understanding the precise mechanism and kinetics of RNA polymerase translocation is crucial for comprehending gene expression and developing therapeutics.

Purpose of the Study:

• To investigate the effect of mechanical force on the translocation rate of T7 RNA polymerase.
• To determine the influence of nucleotide and magnesium ion concentration on the enzyme's catalytic cycle.
• To elucidate the specific step in the catalytic cycle where translocation occurs.

Main Methods:

• Single-molecule assays were employed to measure the translocation rate of T7 RNA polymerase under varying mechanical forces.
• Kinetic parameters were analyzed concerning nucleotide and magnesium ion concentrations.
• Data were integrated with existing biochemical, mutagenic, and crystallographic information.

Main Results:

• Opposing mechanical force acts as a competitive inhibitor of nucleotide binding to the RNA polymerase.
• Magnesium ions appear to be involved in a catalytic step independent of external load force.
• Translocation is proposed to occur after pyrophosphate (PPi) release and before the subsequent nucleotide binding event.

Conclusions:

• The catalytic cycle of RNA polymerase involves translocation after PPi release, not during NTP binding or cleavage.
• Mechanical force and ion concentrations are critical modulators of RNA polymerase activity.
• This research provides a coherent view of the enzyme's mechanism, integrating kinetic and structural data.