Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes - PubMed (original) (raw)

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

P J Rothwell et al. Proc Natl Acad Sci U S A. 2003.

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-A 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.

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Figures

Figure 1

Crystal structure of the RT:dp/dt complex. The protein is displayed as a molecular surface. The different regions of p66 are colored as follows: fingers, purple; palm, yellow; thumb, green; connection, red; and RNase H domain, blue; whereas p51 is colored gray. The nucleic acid substrate is shown as a stick representation with the primer colored purple and the template colored blue. The positions of the labels are shown: the donor dye (Alexa488) is attached at the protein and the acceptor dye (Cy5) is attached at the 5′ end of the primer.

Figure 2

Quantitative single-molecule multiparameter FRET analysis. (Upper) The “species weighted” average lifetimes τ̄D(A) are plotted vs. R DA together with an overlayed curve computed by 6 (τ̄D(0) = 3.1 ns, Φ_FD_ = 0.63, R_0_r = 53 Å). The gray scale indicates an increasing number of bursts. The one-dimensional R DA histograms (magenta color) also display theoretical predictions of single distance distribution profiles (solid lines) and the maximum reasonable R DA value of ≈101 Å for the DA-pair used (dotted lines for donor only species). (Lower) τ̄D(A) is plotted vs. r D together with the overlayed Perrin equation (7) computed for two rotational correlation times ρ_D_ (2 and 7 ns). The value 0.375 is used for _r_0. (A) Donor-labeled RT and Cy5-(5′-dp/dt). Species I is broadly distributed along the line of 6, indicating that it consists of two closely related species (Ia and Ib), which are both mobile and differ slightly in distance, but not in local environment. (B) Donor-labeled RT and unlabeled dp/dt. Only one species is observed, limiting the maximum reasonable value of R DA to 101 Å. (C) Donor-labeled RT and Cy5-(5′-dp/dt) after limited incorporation of four nucleotides. The shifted-species I peak is distributed around a mean distance of 61 Å.

Figure 3

Dissociation of the acceptor-labeled DNA from the donor-labeled RT. Monitoring three characteristic F D/F A ranges for the species in Fig. 2 [species I: 0.7–3.8; species II: 5.7–14.1; species RT:dp/dt (unlabeled): 19.5–291.7], the number of molecules detected in 25-sec intervals is plotted vs. time after a 1,000-fold excess of unlabeled dp/dt was added to the solution. The number of bursts of species I and II decrease with two different characteristic dissociation rate constants k d (6.2 × 10−3 s−1 and 2.6 × 10−3 s−1) accompanied by a corresponding increase in the number of molecules of the trapped species (donor-labeled RT with unlabeled DNA).

Figure 4

(A) Addition of 200 μM dNTPs to a complex of RT:d_dp/dt. The τ̄D(A)-R DA histogram shows only species Ia and II. The productive complex (Ia) interacts with the dp/dt in a state closely resembling the known RT:dp/dt structures. Here, the dNTP is thought to occupy a binding site in the polymerization active site and is, therefore, prebound for incorporation into the primer strand (purple). This state is the productive complex in the educt state (P–E). The solid black lines in the histogram at R DA = 51 Å, and below in the cartoon, indicate the position of the dp/dt bound in the P–E state. The p66 subunit is colored light gray, and the p51 subunit is colored dark gray. The polymerase active site of p66 is colored black. The fluorescence dyes Alexa488 and Cy5 are indicated by balloons colored green and red, respectively. In the P–E state, a dNTP occupies the binding pocket. In the one-dimensional R DA histogram, the distribution of species Ia is observed to be shot-noise limited. (B) Addition of 200 μM sodium pyrophosphate (NaPPi) to a complex of RT:d_dp/dt. The presence of PPi moves the peak toward shorter distances by 5 Å, indicated by species Ib. The presence of PPi shifts the primer terminus into the binding pocket, forming the productive complex in the product state, P–P. The dashed black lines in the histogram at 46 Å, and below in the cartoon, indicate the position of the dp/dt bound in the product state, in the presence of the PPi shifting the dp/dt into the binding cleft. The peak is not Gaussian-distributed, and the rotational correlation time, ρ_D_, remains high. The position of species II remains unchanged, indicating a dead end (DE) complex. Preliminary results indicate that the nucleic acid substrate in the DE complex is bound at a site on the p51 subunit, far removed from the nucleic acid-binding tract observed by crystallography. The cartoon indicates a suggested binding orientation for the DE complex.

References

1. Kohlstaedt L A, Wang J, Friedman J M, Rice P A, Steitz T A. Science. 1992;256:1783–1790. - PubMed
1. Restle T, Müller B, Goody R S. J Biol Chem. 1990;265:8986–8988. - PubMed
1. Restle T, Müller B, Goody R S. FEBS Lett. 1992;300:97–100. - PubMed
1. Sarafianos S G, Das K, Tantillo C, Clark A D, Ding J, Whithcomb J M, Boyer P L, Hughes S H, Arnold E. EMBO J. 2001;20:1449–1461. - PMC - PubMed
1. Jacobo-Molina A, Ding J, Nanni R G, Clark A D, Lu X, Tantillo C, Williams R L, Kamer G, Ferris A L, Clark P, et al. Proc Natl Acad Sci USA. 1993;90:6320–6324. - PMC - PubMed

Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes - PubMed (original) (raw)