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Effects of nucleic acid local structure and magnesium ions on minus-strand transfer mediated by the nucleic acid chaperone activity of HIV-1 nucleocapsid protein.

Wu T, Heilman-Miller SL, Levin JG - Nucleic Acids Res. (2007)

Bottom Line: Using a mutational approach, we show that when the acceptor has a weak local structure, NC has little or no effect.However, when NC is required to destabilize local structure in acceptor RNA, the efficiency of annealing is significantly higher than that of strand transfer.Consistent with this result, we find that Mg2+ (required for RT activity) inhibits NC-catalyzed annealing.

View Article: PubMed Central - PubMed

Affiliation: Section on Viral Gene Regulation, Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone, which is required for highly specific and efficient reverse transcription. Here, we demonstrate that local structure of acceptor RNA at a potential nucleation site, rather than overall thermodynamic stability, is a critical determinant for the minus-strand transfer step (annealing of acceptor RNA to (-) strong-stop DNA followed by reverse transcriptase (RT)-catalyzed DNA extension). In our system, destabilization of a stem-loop structure at the 5' end of the transactivation response element (TAR) in a 70-nt RNA acceptor (RNA 70) appears to be the major nucleation pathway. Using a mutational approach, we show that when the acceptor has a weak local structure, NC has little or no effect. In this case, the efficiencies of both annealing and strand transfer reactions are similar. However, when NC is required to destabilize local structure in acceptor RNA, the efficiency of annealing is significantly higher than that of strand transfer. Consistent with this result, we find that Mg2+ (required for RT activity) inhibits NC-catalyzed annealing. This suggests that Mg2+ competes with NC for binding to the nucleic acid substrates. Collectively, our findings provide new insights into the mechanism of NC-dependent and -independent minus-strand transfer.

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Kinetics of minus-strand transfer with RNA 70 and RNA 70 mutants. Reaction mixtures containing 32P-labeled DNA 50 and acceptor RNA 70 (A), RNA 70U28C (B), RNA 70U30C (C) and RNA 70U28,30C (D), were incubated from 1 to 60 min without NC or with two different NC concentrations (3.5 nt/NC [0.3 µM] and 0.88 nt/NC [1.4 µM]). The percentage (%) of transfer product synthesized was plotted against time of incubation. Symbols: no NC, open circles; 3.5 nt/NC, closed circles; 0.88 nt/NC, closed inverted triangles.
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Figure 4: Kinetics of minus-strand transfer with RNA 70 and RNA 70 mutants. Reaction mixtures containing 32P-labeled DNA 50 and acceptor RNA 70 (A), RNA 70U28C (B), RNA 70U30C (C) and RNA 70U28,30C (D), were incubated from 1 to 60 min without NC or with two different NC concentrations (3.5 nt/NC [0.3 µM] and 0.88 nt/NC [1.4 µM]). The percentage (%) of transfer product synthesized was plotted against time of incubation. Symbols: no NC, open circles; 3.5 nt/NC, closed circles; 0.88 nt/NC, closed inverted triangles.

Mentions: To further investigate whether local structure is a key determinant of NC chaperone activity, we also made three stabilizing mutations in RNA 70 (Figure 2A). It was of interest to determine the effect of these mutations on minus-strand transfer efficiency and in particular, to see whether mutant reactions would become dependent on NC concentration. Here too, our strategy was to test only mutants that have the WT RNA 70-fold. Two point mutations were constructed by changing two G-U wobble pairs to G-C base pairs (bp) (RNA 70U28C or RNA 70U30C). We also made a double mutant by changing both U28 and U30 to C (RNA 70U28,30C), thereby creating two new G-C bp to give a total of four. As expected, the RNA 70 mutants, which have increased stability of local structure, also have higher predicted overall ΔG values than WT (Figure 4, see inserts in each panel).Figure 4.


Effects of nucleic acid local structure and magnesium ions on minus-strand transfer mediated by the nucleic acid chaperone activity of HIV-1 nucleocapsid protein.

Wu T, Heilman-Miller SL, Levin JG - Nucleic Acids Res. (2007)

Kinetics of minus-strand transfer with RNA 70 and RNA 70 mutants. Reaction mixtures containing 32P-labeled DNA 50 and acceptor RNA 70 (A), RNA 70U28C (B), RNA 70U30C (C) and RNA 70U28,30C (D), were incubated from 1 to 60 min without NC or with two different NC concentrations (3.5 nt/NC [0.3 µM] and 0.88 nt/NC [1.4 µM]). The percentage (%) of transfer product synthesized was plotted against time of incubation. Symbols: no NC, open circles; 3.5 nt/NC, closed circles; 0.88 nt/NC, closed inverted triangles.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919501&req=5

Figure 4: Kinetics of minus-strand transfer with RNA 70 and RNA 70 mutants. Reaction mixtures containing 32P-labeled DNA 50 and acceptor RNA 70 (A), RNA 70U28C (B), RNA 70U30C (C) and RNA 70U28,30C (D), were incubated from 1 to 60 min without NC or with two different NC concentrations (3.5 nt/NC [0.3 µM] and 0.88 nt/NC [1.4 µM]). The percentage (%) of transfer product synthesized was plotted against time of incubation. Symbols: no NC, open circles; 3.5 nt/NC, closed circles; 0.88 nt/NC, closed inverted triangles.
Mentions: To further investigate whether local structure is a key determinant of NC chaperone activity, we also made three stabilizing mutations in RNA 70 (Figure 2A). It was of interest to determine the effect of these mutations on minus-strand transfer efficiency and in particular, to see whether mutant reactions would become dependent on NC concentration. Here too, our strategy was to test only mutants that have the WT RNA 70-fold. Two point mutations were constructed by changing two G-U wobble pairs to G-C base pairs (bp) (RNA 70U28C or RNA 70U30C). We also made a double mutant by changing both U28 and U30 to C (RNA 70U28,30C), thereby creating two new G-C bp to give a total of four. As expected, the RNA 70 mutants, which have increased stability of local structure, also have higher predicted overall ΔG values than WT (Figure 4, see inserts in each panel).Figure 4.

Bottom Line: Using a mutational approach, we show that when the acceptor has a weak local structure, NC has little or no effect.However, when NC is required to destabilize local structure in acceptor RNA, the efficiency of annealing is significantly higher than that of strand transfer.Consistent with this result, we find that Mg2+ (required for RT activity) inhibits NC-catalyzed annealing.

View Article: PubMed Central - PubMed

Affiliation: Section on Viral Gene Regulation, Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone, which is required for highly specific and efficient reverse transcription. Here, we demonstrate that local structure of acceptor RNA at a potential nucleation site, rather than overall thermodynamic stability, is a critical determinant for the minus-strand transfer step (annealing of acceptor RNA to (-) strong-stop DNA followed by reverse transcriptase (RT)-catalyzed DNA extension). In our system, destabilization of a stem-loop structure at the 5' end of the transactivation response element (TAR) in a 70-nt RNA acceptor (RNA 70) appears to be the major nucleation pathway. Using a mutational approach, we show that when the acceptor has a weak local structure, NC has little or no effect. In this case, the efficiencies of both annealing and strand transfer reactions are similar. However, when NC is required to destabilize local structure in acceptor RNA, the efficiency of annealing is significantly higher than that of strand transfer. Consistent with this result, we find that Mg2+ (required for RT activity) inhibits NC-catalyzed annealing. This suggests that Mg2+ competes with NC for binding to the nucleic acid substrates. Collectively, our findings provide new insights into the mechanism of NC-dependent and -independent minus-strand transfer.

Show MeSH