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Site-selective probing of cTAR destabilization highlights the necessary plasticity of the HIV-1 nucleocapsid protein to chaperone the first strand transfer.

Godet J, Kenfack C, Przybilla F, Richert L, Duportail G, Mély Y - Nucleic Acids Res. (2013)

Bottom Line: NC(11-55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem.Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G(10) and G(50) the appropriate mobility and accessibility for specific recognition by NC.Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie, UMR 7213 CNRS, Université de Strasbourg, 67401 Illkirch, France.

ABSTRACT
The HIV-1 nucleocapsid protein (NCp7) is a nucleic acid chaperone required during reverse transcription. During the first strand transfer, NCp7 is thought to destabilize cTAR, the (-)DNA copy of the TAR RNA hairpin, and subsequently direct the TAR/cTAR annealing through the zipping of their destabilized stem ends. To further characterize the destabilizing activity of NCp7, we locally probe the structure and dynamics of cTAR by steady-state and time resolved fluorescence spectroscopy. NC(11-55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem. This destabilization is achieved through zinc-finger-dependent binding of NC to the G(10) and G(50) residues. Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G(10) and G(50) the appropriate mobility and accessibility for specific recognition by NC. Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway.

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Quantum yields of the different 2Ap-modified cTAR sequences in the absence and in the presence of NC(11–55). The QY values of the 2-Ap-labelled cTAR sequences were determined at 20°C in the absence (light grey bars) or in the presence of 1 (black bars) or 10 (dark grey bars) equivalents of NC(11–55). In the absence of NC, QY values revealed different base stacking extent within the different cTAR segments. Addition of NC(11–55) differently increased QYs. The strongest effects were observed for positions 9 and 49, suggesting a preferential destabilization in the proximity of these positions.
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gkt164-F2: Quantum yields of the different 2Ap-modified cTAR sequences in the absence and in the presence of NC(11–55). The QY values of the 2-Ap-labelled cTAR sequences were determined at 20°C in the absence (light grey bars) or in the presence of 1 (black bars) or 10 (dark grey bars) equivalents of NC(11–55). In the absence of NC, QY values revealed different base stacking extent within the different cTAR segments. Addition of NC(11–55) differently increased QYs. The strongest effects were observed for positions 9 and 49, suggesting a preferential destabilization in the proximity of these positions.

Mentions: cTAR is an imperfect stem–loop characterized by a 3-nt internal loop, as well as by several bulged nucleotides and mismatches (Figure 1A). We selected labelling positions all along the cTAR Lai sequence. The 2-Ap in position 9 forms a mismatch with C within the lower part of the stem. Positions 28 and 35 are within the central and the internal loop, respectively, whereas positions 53 and 55 are flanking the G54 bulge, and positions 17, 21, 45 and 49 are distributed all over the stem within the different ds-segments. The fluorescence spectra of the different 2-Ap-labelled cTAR sequences exhibited the same maximum emission wavelength (at ∼370 nm) as the free 2-Ap nucleotide. In contrast, their quantum yields (QYs) were strongly reduced in comparison with the free probe (Figure 2), in line with static and dynamic quenching of 2-Ap by its close neighbours in the NAs (52–55). Although QYs were low in all labelled cTAR sequences, clear differences appeared as a function of the labelling positions. QYs were low when 2-Ap was inserted in ds-regions (positions 9, 17, 21, 45, 49, 53 and 55) that are known to favour base stacking, and thus the quenching of its fluorescence (53). The presence of guanine residues neighbouring 2-Ap was associated with a further decrease of the 2-Ap quantum yield, in full line with the most efficient quenching capabilities of Gs among bases (56). On the contrary, the presence of bulges or mismatches in the close vicinity of 2-Ap (positions 9, 53 and 55) was found to lead to somewhat higher QY values (57). By far, the highest QY value was observed when 2-Ap was inserted in the internal loop, suggesting a limited interaction of 2-Ap35 with its neighbours in this single-stranded segment of cTAR. In contrast to 2-Ap35, a significantly lower QY was observed for 2-Ap28, suggesting an efficient quenching of the probe by the G29 residue in the loop. In line with nuclear magnetic resonance (NMR) data showing that the loop of cTAR is highly flexible (58), fast conformational fluctuations may drive 2-Ap28 from unstacked to stacked conformations, resulting in a low QY value. Taken together, we observed a strong dependence of 2-Ap fluorescence signals on the sequence and/or the structure of the DNA segments in which the 2-Ap is inserted. The 2-Ap signals easily discriminated stable regular ds-segments from positions destabilized by bulges or mismatches and clearly showed heterogeneous 2-Ap environments along the cTAR sequence.Figure 2.


Site-selective probing of cTAR destabilization highlights the necessary plasticity of the HIV-1 nucleocapsid protein to chaperone the first strand transfer.

Godet J, Kenfack C, Przybilla F, Richert L, Duportail G, Mély Y - Nucleic Acids Res. (2013)

Quantum yields of the different 2Ap-modified cTAR sequences in the absence and in the presence of NC(11–55). The QY values of the 2-Ap-labelled cTAR sequences were determined at 20°C in the absence (light grey bars) or in the presence of 1 (black bars) or 10 (dark grey bars) equivalents of NC(11–55). In the absence of NC, QY values revealed different base stacking extent within the different cTAR segments. Addition of NC(11–55) differently increased QYs. The strongest effects were observed for positions 9 and 49, suggesting a preferential destabilization in the proximity of these positions.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643577&req=5

gkt164-F2: Quantum yields of the different 2Ap-modified cTAR sequences in the absence and in the presence of NC(11–55). The QY values of the 2-Ap-labelled cTAR sequences were determined at 20°C in the absence (light grey bars) or in the presence of 1 (black bars) or 10 (dark grey bars) equivalents of NC(11–55). In the absence of NC, QY values revealed different base stacking extent within the different cTAR segments. Addition of NC(11–55) differently increased QYs. The strongest effects were observed for positions 9 and 49, suggesting a preferential destabilization in the proximity of these positions.
Mentions: cTAR is an imperfect stem–loop characterized by a 3-nt internal loop, as well as by several bulged nucleotides and mismatches (Figure 1A). We selected labelling positions all along the cTAR Lai sequence. The 2-Ap in position 9 forms a mismatch with C within the lower part of the stem. Positions 28 and 35 are within the central and the internal loop, respectively, whereas positions 53 and 55 are flanking the G54 bulge, and positions 17, 21, 45 and 49 are distributed all over the stem within the different ds-segments. The fluorescence spectra of the different 2-Ap-labelled cTAR sequences exhibited the same maximum emission wavelength (at ∼370 nm) as the free 2-Ap nucleotide. In contrast, their quantum yields (QYs) were strongly reduced in comparison with the free probe (Figure 2), in line with static and dynamic quenching of 2-Ap by its close neighbours in the NAs (52–55). Although QYs were low in all labelled cTAR sequences, clear differences appeared as a function of the labelling positions. QYs were low when 2-Ap was inserted in ds-regions (positions 9, 17, 21, 45, 49, 53 and 55) that are known to favour base stacking, and thus the quenching of its fluorescence (53). The presence of guanine residues neighbouring 2-Ap was associated with a further decrease of the 2-Ap quantum yield, in full line with the most efficient quenching capabilities of Gs among bases (56). On the contrary, the presence of bulges or mismatches in the close vicinity of 2-Ap (positions 9, 53 and 55) was found to lead to somewhat higher QY values (57). By far, the highest QY value was observed when 2-Ap was inserted in the internal loop, suggesting a limited interaction of 2-Ap35 with its neighbours in this single-stranded segment of cTAR. In contrast to 2-Ap35, a significantly lower QY was observed for 2-Ap28, suggesting an efficient quenching of the probe by the G29 residue in the loop. In line with nuclear magnetic resonance (NMR) data showing that the loop of cTAR is highly flexible (58), fast conformational fluctuations may drive 2-Ap28 from unstacked to stacked conformations, resulting in a low QY value. Taken together, we observed a strong dependence of 2-Ap fluorescence signals on the sequence and/or the structure of the DNA segments in which the 2-Ap is inserted. The 2-Ap signals easily discriminated stable regular ds-segments from positions destabilized by bulges or mismatches and clearly showed heterogeneous 2-Ap environments along the cTAR sequence.Figure 2.

Bottom Line: NC(11-55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem.Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G(10) and G(50) the appropriate mobility and accessibility for specific recognition by NC.Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie, UMR 7213 CNRS, Université de Strasbourg, 67401 Illkirch, France.

ABSTRACT
The HIV-1 nucleocapsid protein (NCp7) is a nucleic acid chaperone required during reverse transcription. During the first strand transfer, NCp7 is thought to destabilize cTAR, the (-)DNA copy of the TAR RNA hairpin, and subsequently direct the TAR/cTAR annealing through the zipping of their destabilized stem ends. To further characterize the destabilizing activity of NCp7, we locally probe the structure and dynamics of cTAR by steady-state and time resolved fluorescence spectroscopy. NC(11-55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem. This destabilization is achieved through zinc-finger-dependent binding of NC to the G(10) and G(50) residues. Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G(10) and G(50) the appropriate mobility and accessibility for specific recognition by NC. Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway.

Show MeSH
Related in: MedlinePlus