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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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Effect of Mg2+ on annealing of RNA 70 and RNA 70 mutants to DNA 50. 32P-labeled DNA 50 was incubated with RNA 70 (lanes 1–8), RNA 70G53,54A (lanes 9–16) or RNA 70U28,30C (lanes 17–24) for 30 min at 37°C under conditions indicated below. Bar graphs show the percentage (%) of DNA 50 annealed for each reaction. Symbols: open bars, no NC and no Mg2+ (lanes 1, 9, 17); striped bars, 7 mM Mg2+ alone (lanes 2, 10, 18); gray bars, 0.88 nt/NC (1.4 µM) and no Mg2+ (lanes 3, 11, 19); black bars, 0.88 nt/NC and increasing concentrations of Mg2+as follows: 0.25 mM Mg2+ (lanes 4, 12, 20); 0.5 mM Mg2+ (lanes 5, 13, 21); 1.75 mM Mg2+ (lanes 6, 14, 22); 3.5 mM Mg2+ (lanes 7, 15, 23); 7 mM Mg2+ (lanes 8, 16, 24). Note that the order of addition of NC and MgCl2 had no effect on the extent of annealing (data not shown).
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Figure 7: Effect of Mg2+ on annealing of RNA 70 and RNA 70 mutants to DNA 50. 32P-labeled DNA 50 was incubated with RNA 70 (lanes 1–8), RNA 70G53,54A (lanes 9–16) or RNA 70U28,30C (lanes 17–24) for 30 min at 37°C under conditions indicated below. Bar graphs show the percentage (%) of DNA 50 annealed for each reaction. Symbols: open bars, no NC and no Mg2+ (lanes 1, 9, 17); striped bars, 7 mM Mg2+ alone (lanes 2, 10, 18); gray bars, 0.88 nt/NC (1.4 µM) and no Mg2+ (lanes 3, 11, 19); black bars, 0.88 nt/NC and increasing concentrations of Mg2+as follows: 0.25 mM Mg2+ (lanes 4, 12, 20); 0.5 mM Mg2+ (lanes 5, 13, 21); 1.75 mM Mg2+ (lanes 6, 14, 22); 3.5 mM Mg2+ (lanes 7, 15, 23); 7 mM Mg2+ (lanes 8, 16, 24). Note that the order of addition of NC and MgCl2 had no effect on the extent of annealing (data not shown).

Mentions: The apparent increased efficiency of the annealing reaction with the 5′ stem-loop mutants compared with their activity in strand transfer was unexpected. We wondered whether the absence of RT, dNTPs, and 7 mM MgCl2 in the annealing reaction mixtures was responsible for this result. Addition of RT or dNTPs, either singly or in combination, to annealing reactions with RNA 70, RNA 70G53,54A, or RNA 70U28,30 did not change the level of annealing (data not shown). In contrast, when increasing concentrations of Mg2+ were added, there was an effect on RNA–DNA hybrid formation (Figure 7).Figure 7.


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)

Effect of Mg2+ on annealing of RNA 70 and RNA 70 mutants to DNA 50. 32P-labeled DNA 50 was incubated with RNA 70 (lanes 1–8), RNA 70G53,54A (lanes 9–16) or RNA 70U28,30C (lanes 17–24) for 30 min at 37°C under conditions indicated below. Bar graphs show the percentage (%) of DNA 50 annealed for each reaction. Symbols: open bars, no NC and no Mg2+ (lanes 1, 9, 17); striped bars, 7 mM Mg2+ alone (lanes 2, 10, 18); gray bars, 0.88 nt/NC (1.4 µM) and no Mg2+ (lanes 3, 11, 19); black bars, 0.88 nt/NC and increasing concentrations of Mg2+as follows: 0.25 mM Mg2+ (lanes 4, 12, 20); 0.5 mM Mg2+ (lanes 5, 13, 21); 1.75 mM Mg2+ (lanes 6, 14, 22); 3.5 mM Mg2+ (lanes 7, 15, 23); 7 mM Mg2+ (lanes 8, 16, 24). Note that the order of addition of NC and MgCl2 had no effect on the extent of annealing (data not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 7: Effect of Mg2+ on annealing of RNA 70 and RNA 70 mutants to DNA 50. 32P-labeled DNA 50 was incubated with RNA 70 (lanes 1–8), RNA 70G53,54A (lanes 9–16) or RNA 70U28,30C (lanes 17–24) for 30 min at 37°C under conditions indicated below. Bar graphs show the percentage (%) of DNA 50 annealed for each reaction. Symbols: open bars, no NC and no Mg2+ (lanes 1, 9, 17); striped bars, 7 mM Mg2+ alone (lanes 2, 10, 18); gray bars, 0.88 nt/NC (1.4 µM) and no Mg2+ (lanes 3, 11, 19); black bars, 0.88 nt/NC and increasing concentrations of Mg2+as follows: 0.25 mM Mg2+ (lanes 4, 12, 20); 0.5 mM Mg2+ (lanes 5, 13, 21); 1.75 mM Mg2+ (lanes 6, 14, 22); 3.5 mM Mg2+ (lanes 7, 15, 23); 7 mM Mg2+ (lanes 8, 16, 24). Note that the order of addition of NC and MgCl2 had no effect on the extent of annealing (data not shown).
Mentions: The apparent increased efficiency of the annealing reaction with the 5′ stem-loop mutants compared with their activity in strand transfer was unexpected. We wondered whether the absence of RT, dNTPs, and 7 mM MgCl2 in the annealing reaction mixtures was responsible for this result. Addition of RT or dNTPs, either singly or in combination, to annealing reactions with RNA 70, RNA 70G53,54A, or RNA 70U28,30 did not change the level of annealing (data not shown). In contrast, when increasing concentrations of Mg2+ were added, there was an effect on RNA–DNA hybrid formation (Figure 7).Figure 7.

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
Related in: MedlinePlus