Substrate unwinding by Reverse Transcriptase: Lessons from single-molecule studies

Ariel Kaplan, Faculty of Biology, Technion - Israel Institute of Technology, Haifa, Israel

Retroviral Reverse Transcriptase (RT) is a multi-functional enzyme that catalyzes the synthesis of an integration-competent dsDNA molecule, using as a substrate the viral RNA. During polymerization, RT is able to overcome structural barriers in the form of RNA secondary structures (e.g hairpins) by itself, without external unwinding proteins such as helicases. Previous studies have shown that polymerization rates during “strand displacement” polymerization are slower, suggesting that secondary structure motifs may serve to regulate RT’s activity. However, the mechanism of nucleic-acid unwinding by RT, and its coupling with polymerization, remain poorly understood.  In our research we use a single-molecule optical-tweezers approach to study the polymerization activity of Moloney Murine Leukemia Virus RT on a DNA hairpin template, as a function of the tension applied on the hairpin and the concentration of dNTPs. We find that polymerization rate differences between high and low forces are significantly larger as the dNTPs concentration increases. This interplay between concentration and force suggests a “passive” model of the RT polymerization. In addition, measuring the Michaelis-Menten parameters at different forces allow us to locate the translocation step of the enzyme and brings us one step closer to elucidating its full mechano-chemical cycle.