Single-molecule FRET studies of important intermediates in the nucleocapsid-protein-chaperoned minus-strand transfer step in HIV-1 reverse transcription

Abstract

The minus-strand transfer step of HIV-1 reverse transcription is chaperoned by the nucleocapsid protein (NC), which has been shown to facilitate the annealing between the transactivation response element (TAR) RNA and complementary TAR DNA stem-loop structures. In this work, potential intermediates in the mechanism of NC-chaperoned TAR DNA/TAR RNA annealing have been examined using single-molecule fluorescence resonance energy transfer. The interaction between TAR DNA and various DNA oligonucleotides designed to mimic the initial annealing step was monitored to capture potential intermediates along the reaction pathway. Two possible mechanisms of annealing were examined, namely nucleation through the 3'/5' termini, termed the "zipper" complex, or nucleation through the hairpin loops in a "kissing" complex. Intermediates associated with both mechanisms were observed in the presence of NC, and the kinetics of formation of these intermediates were also measured. Thus, the single-molecule experiments support the notion that NC-assisted annealing of TAR DNA:TAR RNA may occur through multiple pathways.

SCHEME 1

Previously, SMFRET was used to demonstrate that, in the presence of NC, the TAR DNA hairpin is “open” through the terminal two-loop regions (the four loops are indicated above by L1–L4) (41). The partially open form that exists in the presence of NC is termed the Y Form.

SCHEME 2

The two intermediates in the NC-chaperoned annealing of TAR DNA and TAR RNA that are examined in this study are illustrated. (Left) A zipper mechanism can occur if annealing initiates through the 3′ or 5′ terminus (or both simultaneously, as shown in the case of a Y:Y complex). (Right) A kissing mechanism occurs if annealing is initiated through interaction between the bottom loop regions.

SCHEME 3

Proposed secondary structure of TAR DNA (right) and sequences of complementary and noncomplementary DNA oligonucleotides used in this study (left) are shown. (Right) Region A, B, C, and D represent the regions complementary to the DNA oligonucleotides shown on the left. (Left) Oligonucleotides preceded by “A” are Cy5 labeled. A-13-N and 12-N are oligonucleotides designed to be noncomplementary to TAR DNA, as defined by fewer than four consecutive Watson-Crick basepairs.

FIGURE 1

Ensemble E_A values of doubly labeled TAR DNA at three different τ_B under different reaction conditions are shown. (Column 1) E_A histogram before the addition of NC. (Column 2) E_A histogram after addition of 445 nM NC. (Column 3) E_A in the presence of 445 nM NC and 100 nM noncomplementary 12-N oligomer. (Column 4) E_A in the presence of 445 nM NC and 100 nM complementary 12-C oligomer. All experiments were performed at 2 mM Mg²⁺.

FIGURE 2

A summary of the reaction between doubly labeled TAR DNA and oligonucleotide 12-C at two concentrations is shown. The ensemble distributions of E_A were obtained at 2 mM Mg²⁺, 445 nM NC, and 10 nM 12-C (top) or 100 nM 12-C (bottom). At higher [12-C], the structure of TAR is shifted from Y states to bound states. The data are presented using τ_B = 10 ms.

FIGURE 3

(A–E) Ensemble E_A histograms (middle) and representative single-molecule E_A time trajectories (right) are shown for the reactions shown at left. All measurements were performed using donor-labeled TAR DNA and acceptor-labeled oligonucleotides in the presence of 445 nM NC and 2 mM Mg²⁺. The data are presented using τ_B = 50 ms. The oligonucleotides used are: (A) 10 nM A-27-A, (B) 10 nM A-24-B. The inset shows that no observable binding occurs in a minor population of the single molecules measured. (C) For 100 nM A-13-C, the distributions of E_A and the E_A trajectory exhibit reversibility. (D) Similar reversible binding was observed with A-14-D, and (E) 100 nM A13-N. No binding was observed for 100 nM A-13-N.

FIGURE 4

Kinetic curves for the annealing rates of the zipper mimic, A-27-A, versus the kissing mimic, A-24-B, with donor-labeled TAR DNA. These experiments were performed in the presence of 445 nM NC and 2 mM Mg²⁺. The oligonucleotide concentrations were 10 nM in both cases. Analogous reactions with inverted donor-labeled TAR DNA are also included in the figure.

FIGURE 5

(A) An ensemble E_A histogram is shown for the simultaneous reaction of A-27-A and A-24-B with donor-labeled TAR DNA at [Mg²⁺] = 2 mM and [NC] = 445 nM. The 〈E_A〉 of 0.92 is consistent with A-27-A binding. (B) An ensemble E_A histogram for the reaction of 23-A and A-24-B with donor-labeled TAR DNA under similar reaction conditions results in an 〈E_A〉 of 0.75, consistent with A-24-B binding. The data are presented using τ_B = 10 ms.