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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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Annealing of RNA 70 and mutants to DNA 50. 32P-labeled DNA 50 was incubated with RNA 70 (A), RNA 70G53,54A (B), RNA 70U28C (C) and RNA 70U28,30C (D) from 0.5 to 30 min at 37°C 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 DNA 50 annealed was plotted against time of incubation. Symbols are the same as those given in Figure 4 legend.
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Figure 6: Annealing of RNA 70 and mutants to DNA 50. 32P-labeled DNA 50 was incubated with RNA 70 (A), RNA 70G53,54A (B), RNA 70U28C (C) and RNA 70U28,30C (D) from 0.5 to 30 min at 37°C 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 DNA 50 annealed was plotted against time of incubation. Symbols are the same as those given in Figure 4 legend.

Mentions: To gain a further understanding of the mechanism of NC chaperone activity during minus-strand transfer, it is important to know how changes in local structure affect the kinetics of annealing. The annealing reaction leads to formation of an RNA-DNA hybrid containing complementary R sequences in acceptor RNA and (-) SSDNA (3). In this study, the experiments were performed with WT RNA 70 and RNA 70 mutants (Figure 6). The results are expressed as the percentage of 32P-labeled DNA 50 annealed in the reaction.Figure 6.


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)

Annealing of RNA 70 and mutants to DNA 50. 32P-labeled DNA 50 was incubated with RNA 70 (A), RNA 70G53,54A (B), RNA 70U28C (C) and RNA 70U28,30C (D) from 0.5 to 30 min at 37°C 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 DNA 50 annealed was plotted against time of incubation. Symbols are the same as those given in Figure 4 legend.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Annealing of RNA 70 and mutants to DNA 50. 32P-labeled DNA 50 was incubated with RNA 70 (A), RNA 70G53,54A (B), RNA 70U28C (C) and RNA 70U28,30C (D) from 0.5 to 30 min at 37°C 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 DNA 50 annealed was plotted against time of incubation. Symbols are the same as those given in Figure 4 legend.
Mentions: To gain a further understanding of the mechanism of NC chaperone activity during minus-strand transfer, it is important to know how changes in local structure affect the kinetics of annealing. The annealing reaction leads to formation of an RNA-DNA hybrid containing complementary R sequences in acceptor RNA and (-) SSDNA (3). In this study, the experiments were performed with WT RNA 70 and RNA 70 mutants (Figure 6). The results are expressed as the percentage of 32P-labeled DNA 50 annealed in the reaction.Figure 6.

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