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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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Schematic diagram illustrating the strand transfer system used for this study. (A) RNA 70. The diagram shows annealing of the 50 complementary bases in the RNA 70 acceptor and the (−) SSDNA, DNA 50, which is labeled at its 5′ end with 32P. The 20 nt sequence from U3 serves as the template for RT-catalyzed DNA extension. (B) RNA 50. The only difference between (A) and (B) is that the RNA 50 acceptor contains only 30 nt complementary to DNA 50. With both acceptors, the final product is a 70-nt labeled DNA (dashed lines at the bottom of the figure). For RNA 70, the ‘TAR’ sequence is 50 nt; 9 nt at the 3′ end of full-length TAR are missing (28). RNA 50 ‘TAR’ consists of the 5′ half of TAR (28). White rectangles, DNA 50; gray rectangles, acceptor RNA. The stars denote the 32P label. The diagram is not drawn to scale.
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Figure 1: Schematic diagram illustrating the strand transfer system used for this study. (A) RNA 70. The diagram shows annealing of the 50 complementary bases in the RNA 70 acceptor and the (−) SSDNA, DNA 50, which is labeled at its 5′ end with 32P. The 20 nt sequence from U3 serves as the template for RT-catalyzed DNA extension. (B) RNA 50. The only difference between (A) and (B) is that the RNA 50 acceptor contains only 30 nt complementary to DNA 50. With both acceptors, the final product is a 70-nt labeled DNA (dashed lines at the bottom of the figure). For RNA 70, the ‘TAR’ sequence is 50 nt; 9 nt at the 3′ end of full-length TAR are missing (28). RNA 50 ‘TAR’ consists of the 5′ half of TAR (28). White rectangles, DNA 50; gray rectangles, acceptor RNA. The stars denote the 32P label. The diagram is not drawn to scale.

Mentions: In a previous study of NC chaperone activity and minus-strand transfer (28), a series of acceptor RNAs truncated in U3, the 3′ region of R, and TAR, were assayed with (−) SSDNAs having comparable truncations in complementary sequences, except that a portion of U5 rather than U3 was deleted. In general, in assays with the same DNA, acceptor RNAs with low predicted free energies of folding had more strand transfer activity than more highly structured acceptors. However, we found one striking exception when two acceptor RNAs, RNA 70 and RNA 50, were assayed with the DNA 50 (−) SSDNA (see below). To determine whether this finding could lead to new insights regarding the mechanism of nucleic acid chaperone activity in minus-strand transfer, we used the model system illustrated in Figure 1. The figure shows annealing of RNA 70 (A) or RNA 50 (B) to DNA 50 as well as the 20-nt U3 RNA sequence, which serves as the template for RT-catalyzed extension of DNA 50 to a 70-nt product.


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)

Schematic diagram illustrating the strand transfer system used for this study. (A) RNA 70. The diagram shows annealing of the 50 complementary bases in the RNA 70 acceptor and the (−) SSDNA, DNA 50, which is labeled at its 5′ end with 32P. The 20 nt sequence from U3 serves as the template for RT-catalyzed DNA extension. (B) RNA 50. The only difference between (A) and (B) is that the RNA 50 acceptor contains only 30 nt complementary to DNA 50. With both acceptors, the final product is a 70-nt labeled DNA (dashed lines at the bottom of the figure). For RNA 70, the ‘TAR’ sequence is 50 nt; 9 nt at the 3′ end of full-length TAR are missing (28). RNA 50 ‘TAR’ consists of the 5′ half of TAR (28). White rectangles, DNA 50; gray rectangles, acceptor RNA. The stars denote the 32P label. The diagram is not drawn to scale.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Schematic diagram illustrating the strand transfer system used for this study. (A) RNA 70. The diagram shows annealing of the 50 complementary bases in the RNA 70 acceptor and the (−) SSDNA, DNA 50, which is labeled at its 5′ end with 32P. The 20 nt sequence from U3 serves as the template for RT-catalyzed DNA extension. (B) RNA 50. The only difference between (A) and (B) is that the RNA 50 acceptor contains only 30 nt complementary to DNA 50. With both acceptors, the final product is a 70-nt labeled DNA (dashed lines at the bottom of the figure). For RNA 70, the ‘TAR’ sequence is 50 nt; 9 nt at the 3′ end of full-length TAR are missing (28). RNA 50 ‘TAR’ consists of the 5′ half of TAR (28). White rectangles, DNA 50; gray rectangles, acceptor RNA. The stars denote the 32P label. The diagram is not drawn to scale.
Mentions: In a previous study of NC chaperone activity and minus-strand transfer (28), a series of acceptor RNAs truncated in U3, the 3′ region of R, and TAR, were assayed with (−) SSDNAs having comparable truncations in complementary sequences, except that a portion of U5 rather than U3 was deleted. In general, in assays with the same DNA, acceptor RNAs with low predicted free energies of folding had more strand transfer activity than more highly structured acceptors. However, we found one striking exception when two acceptor RNAs, RNA 70 and RNA 50, were assayed with the DNA 50 (−) SSDNA (see below). To determine whether this finding could lead to new insights regarding the mechanism of nucleic acid chaperone activity in minus-strand transfer, we used the model system illustrated in Figure 1. The figure shows annealing of RNA 70 (A) or RNA 50 (B) to DNA 50 as well as the 20-nt U3 RNA sequence, which serves as the template for RT-catalyzed extension of DNA 50 to a 70-nt product.

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