Step-by-step progress toward understanding the hepatitis C virus RNA helicase

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Helicases are crucial enzymes in DNA and RNA metabolism.
• Understanding their ATP-driven mechanisms is key to cellular function.
• Previous studies lacked the resolution to observe individual enzyme cycles.

Purpose of the Study:

• To visualize the real-time RNA translocation and unwinding by a hepatitis C virus helicase (NS3).
• To elucidate the detailed mechanism coupling ATP hydrolysis to nucleic acid rearrangement.
• To provide insights into the function of superfamily-2 helicases.

Main Methods:

• Real-time observation of NS3 helicase activity at high resolution (2 base pairs, 20 ms).
• Tracking RNA translocation and unwinding cycles of the NS3 monomer.
• Utilizing a novel assay for studying nucleic acid translocation motors.

Main Results:

• NS3 helicase moves in discrete ATP-dependent steps of 11 +/- 3 base pairs.
• RNA unwinding occurs in rapid substeps of 3.6 +/- 1.3 base pairs, triggered by ATP binding.
• The NS3 helicase exhibits an inchworm-like movement mechanism.

Conclusions:

• The NS3 helicase couples ATP binding and hydrolysis to stepwise RNA translocation and unwinding.
• This inchworm-like mechanism is likely conserved in other non-hexameric helicases.
• The developed assay is valuable for studying various nucleic acid motor proteins.