Single-Molecule Analysis of Reverse Transcriptase Enzymes

Abstract

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. Outline 1 Introduction 2 Implications of RT discoveries 3 On the evolution of viral RTs and telomerase4 Single-molecule methods to study RT enzymes 5 Human immunodeficiency virus (HIV) RT 6 Conformational dynamics of HIV RT on nucleic acids 7 RT sliding, substrate recognition, and strand displacement synthesis 8 Structural dynamics of the HIV RT initiation complex 9 Telomerase 10 Structural dynamics of Tetrahymena thermophila telomerase 11 Nucleic acid dynamics during human telomerase catalysis 12 Conclusions and perspectives References

Sliding of HIV-1 reverse transcriptase over DNA creates a transient P pocket – Targeting P-pocket by fragment screening

“…The dynamic behavior of RT ipping and sliding over a dsDNA or an RNA/DNA substrate have been observed by single-molecule FRET studies. 14,15,26 Our current crystallization requires Cd 2+ ions and the structure revealed that two active site Mg 2+ ions in the N complex have been replaced by two Cd 2+ ions (Fig. 1f); signi cantly strong scatterer Cd 2+ ions that has 46 electrons compared to Mg 2+ ions that has 10 electrons produced strong electron density peaks;…”

“…In general, the transient states have been considered as important targets for drug design 13 , and there may exist unidenti ed transient states of HIV-1 RT that can be valuable for discovering new classes of drugs. During the polymerization process, RT has been shown to slide over its substrate by singlemolecule FRET studies, 14,15 which indicates potential for the existence of transient states, however, no transient state has been structurally characterized. In this study, we crystallized HIV-1 RT/dsDNA in a new crystal form with two copies of the complex present in the crystallographic asymmetric unit; in one, the primer 3'-end nucleotide occupies the N site representing the pre-translocation complex (N complex) and in the other copy, RT slides ahead of dsDNA such that the primer 3' terminus occupies P-1 site (P-1 complex).…”

Sliding of HIV-1 reverse transcriptase over DNA creates a transient P pocket – Targeting P-pocket by fragment screening

“…The dynamic behavior of RT flipping and sliding over a dsDNA or an RNA/DNA substrate have been observed by single-molecule FRET studies. 14,15,26 Our current crystallization requires Cd 2+ ions and the structure revealed that two active site Mg 2+ ions in the N complex have been replaced by two Cd 2+ ions (Fig. 1f); significantly strong scatterer Cd 2+ ions that has 46 electrons compared to Mg 2+ ions that has 10 electrons produced strong electron density peaks; Cd 2+ ions were unambiguously located in the structure.…”

“…In general, the transient states have been considered as important targets for drug design 13 , and there may exist unidentified transient states of HIV-1 RT that can be valuable for discovering new classes of drugs. During the polymerization process, RT has been shown to slide over its substrate by single-molecule FRET studies, 14,15 which indicates potential for the existence of transient states, however, no transient state has been structurally characterized. In this study, we crystallized HIV-1 RT/dsDNA in a new crystal form with two copies of the complex present in the crystallographic asymmetric unit; in one, the primer 3’-end nucleotide occupies the N site representing the pre-translocation complex (N complex) and in the other copy, RT slides ahead of dsDNA such that the primer 3’ terminus occupies P-1 site (P-1 complex).…”

Crystal structures of N-terminally truncated telomerase reverse transcriptase from fungi

“…The addition of consecutive copies of telomeric repeats involves extremely complex and delicate processes, including dissociation, translocation and realignment of the RNA-DNA hybrid ( 31 ). Telomerase must undergo extensive domain or motif rearrangements for each round of telomere addition to accomplish these tasks ( 58 ). However, such structural mobility remains speculative and direct structural evidence is still lacking ( 12 ).…”