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

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.H IV-1 reverse transcriptase (RT) is a key enzyme in the life cycle of HIV. This multifunctional enzyme is responsible for the complex process of transcribing viral RNA into doublestranded DNA for integration into the host cell genome. The enzyme is a heterodimer composed of subunits which share a common N terminus and have subdomains referred to as fingers, palm, thumb, and connection ( Fig. 1). Although the subdomains of each subunit are structurally similar, the overall folding within the two subunits is quite different (1). The large subunit, p66, contains RNA-and DNA-dependent DNA polymerase as well as RNase H activities. The p51 subunit, which is inactive in the heterodimer but active in homodimers (2, 3), is thought to play a role in stabilizing the conformation of p66.Crystallographic studies of RT:nucleic acid primer͞template (p͞t) complexes have provided valuable insights into the structural features and conformational changes induced by p͞t binding (1, 4-7). To date, these crystallographic models have indicated a single p͞t-binding mode. In contrast, recent solutionbased kinetic studies on p͞t binding and nucleotide incorporation by RT suggest a heterogeneous mixture of several different binding modes (8). To confirm directly the existence of several species, and to obtain structural and functional information about each, we have used single-molecule spectroscopy to investigate RT:p͞t complexes in solution.Single-molecule techniques have proven to be valuable tools for resolving static and dynamic heterogeneity of an ensemble (9-13). For this investigation, we use a newly developed multiparameter fluorescence detection (MF...

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