Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes

^‡Abteilung Spektroskopie und Photochemische Kinetik, Max-Planck-Institut für Biophysikalische Chemie, Am Fassberg 11, D-37077 Göttingen, Germany; and ^*Abteilung Physikalische Biochemie, Max-Planck-Institut für Molekulare Physiologie, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany

Edited by James A. Spudich, Stanford University School of Medicine, Stanford, CA, and approved December 23, 2002 (received for review July 5, 2002)

Abstract

By using single-molecule multiparameter fluorescence detection, fluorescence resonance energy transfer experiments, and newly developed data analysis methods, this study demonstrates directly the existence of three structurally distinct forms of reverse transcriptase (RT):nucleic acid complexes in solution. Single-molecule multiparameter fluorescence detection also provides first information on the structure of a complex not observed by x-ray crystallography. This species did not incorporate nucleotides and is structurally distinct from the other two observed species. We determined that the nucleic acid substrate is bound at a site far removed from the nucleic acid-binding tract observed by crystallography. In contrast, the other two states are identified as being similar to the x-ray crystal structure and represent distinct enzymatically productive stages in DNA polymerization. These species differ by only a 5-Å shift in the position of the nucleic acid. Addition of nucleoside triphosphate or of inorganic pyrophosphate allowed us to assign them as the educt and product state in the polymerization reaction cycle; i.e., the educt state is a complex in which the nucleic acid is positioned to allow nucleotide incorporation. The second RT:nucleic acid complex is the product state, which is formed immediately after nucleotide incorporation, but before RT translates to the next nucleotide.

Footnotes

↵ † P.J.R., S.B., and O.K. contributed equally to this work.
↵ § Present address: Solvias AG Klybeckstrasse 191, Postfach, CH-4002 Basel, Switzerland.
↵ ¶ Present address: DIREVO Biotech AG, Biochemical Assay Development, Nattermannallee 1, D-50829 Köln, Germany.
↵ ‖ To whom correspondence should be addressed. E-mail: roger.goody{at}mpi-dortmund.mpg.de or cseidel{at}gwdg.de.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

RT,

reverse transcriptase;

p/t,

primer/template;

MFD,

multiparameter fluorescence detection;

F,

fluorescence intensity;

τ,

lifetime;

r,

anisotropy;

t,

time;

λ,

spectral windows;

FRET,

fluorescence resonance energy transfer;

DE,

dead-end complex;

A,

acceptor fluorophore;

D,

donor fluorophore;

dNTP,

deoxy-nucleoside triphosphate;

dp/dt,

DNA primer/DNA template;

RDA,

donor-acceptor distance;

sMFD,

single-molecule MFD