Ph.D Student | Malik Omri |
---|---|
Subject | The Mechano-Chemistry of Reverse Transcriptase |
Department | Department of Nanoscience and Nanotechnology | Supervisor | Professor Ariel Kaplan |
Reverse transcriptase (RT) catalyzes the conversion of retroviral
RNA into an integration-competent double-stranded DNA , with a variety of
enzymatic activities that include “strand displacement” DNA synthesis, i.e. the
ability to unwind nucleic-acids duplexes concomitantly with polymerization. A comprehensive
understanding of strand displacement synthesis by RT and its potential
regulation by secondary structure motifs is lacking, in part due to the
limitations of traditional biochemical techniques. In our research, we designed
and constructed a high-resolution optical-tweezers to study the polymerization
activity of the Moloney Murine Leukemia Virus RT
on a DNA hairpin template, at the single molecule level and on a wide range of
chemical and mechanical conditions. Our results show that strand-displacement
polymerization is frequently interrupted by inactive events, which include
intrinsic pauses during which the enzyme remains bound to the template and
events of enzyme dissociation followed by reinitiation. The pauses were
shown to be modulated by the strength of the DNA duplex ∼8 bp ahead, indicating the existence of uncharacterized RT/DNA
interactions, and correspond to backtracking of the enzyme, whose recovery is
also modulated by the duplex strength. Dissociation and reinitiation events,
which induce long periods of inactivity and are likely the rate-limiting step
in the synthesis of the genome in vivo, are modulated by the template structure
and the viral nucleocapsid protein. Moreover, our results
indicate that RT functions as a Brownian ratchet, with dNTP binding as the rectifying
reaction of the ratchet. We also found that RT is a relatively passive enzyme,
able to polymerize on structured templates only by exploiting their thermal
breathing. Taken
together, our results elucidate the mechanism of polymerization by RT and emphasize
the potential regulatory role of conserved structural motifs, which may provide
useful information for the development of better inhibitors to function as
anti-retroviral drugs. Finally, to further explore the process of reverse
transcription, in particular the interplay between the enzyme’s function and
the elongation complex structure, we designed and built a new and versatile
instrument, Fleezers, that uses acousto-optic
modulation combineto
combines high-resolution optical tweezers with
different modalities of single-molecule
fluorescence detection. As a
proof-of-concept
of the integrated system, we used
the Fleezers to
stretch a
DNA tether
and
applied epi-fluorescence
imaging and
confocal detection to monitor the
fluorescence of a
trapped fluorescently-labeled
microspheres. These
demonstrations have
indicated the Fleezers feasibility
to simultaneously
study RT
function-structure relationships
and its interaction with
other viral or cellular
factors by multipole single-molecule
techniques.