טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentMalik Omri
SubjectMechanism of Secondary Structure Unwinding by
Reverse Transcriptase
DepartmentDepartment of Physics
Supervisor Professor Ariel Kaplan


Abstract

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. Following the stochastic nature of RT and its base-pair scale dynamics challenged conventional bulk biochemical methods. Moreover, RT is a molecular motor, i.e., it converts chemical energy to mechanical work. Hence, the ability to apply an external force to inhibit or facilitate polymerization is an essential tool to dissect RT’s mechanism. In our research we developed and constructed a single-molecule high resolution optical-tweezers to study the polymerization activity of Moloney Murine Leukemia Virus RT on a DNA hairpin template. The instrumental, environmental and brownian noise were thoroughly reduced and we demonstrated that our system is capable of sensing extension changes of a single base pair. Calibration of the system enabled us to follow the strand displacement DNA polymerization of the DNA hairpin as a function of its applied tension and the concentration of dNTPs.