Single-Molecule Analysis of Reverse Transcriptase Enzymes.

The original discovery of enzymes that synthesize DNA using an RNA template appeared to contradict the central dogma of biology, in which information is transferred, in a unidirectional way, from DNA genes into RNA molecules. The paradigm-shifting discovery of RNA-dependent DNA polymerases, also called reverse transcriptases (RTs), reshaped existing views for how cells function; however, the scope of the impact RTs impose on biology had yet to be realized. In the decades of research since the early 1970s, the biomedical and biotechnological significance of retroviral RTs, as well as the evolutionarily related telomerase enzyme, has become exceedingly clear. One common theme that has emerged in the course of RT-related research is the central role of nucleic acid binding and dynamics during enzyme function. However, directly interrogating these dynamic properties is challenging because of the stochastic properties of biological macromolecules. In this review, we describe how the development of single-molecule biophysical techniques has opened new windows through which to observe the dynamic behavior of this remarkable class of enzymes. Specifically, we focus on how the powerful single-molecule Förster resonance energy transfer (FRET) method has been exploited to study the structure and function of the human immunodeficiency virus (HIV) RT and telomerase ribonucleoprotein (RNP) enzymes. These exciting studies have refined our understanding of RT catalysis, have revealed unforeseen structural rearrangements between RTs and their nucleic acid substrates, and have helped to characterize the mode of action of RT-inhibiting drugs. We conclude with a discussion of how the ongoing development of single-molecule technologies will continue to empower researchers to probe RT mechanisms in new and exciting ways.