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The RNA annealing mechanism of the HIV-1 Tat peptide: conversion of the RNA into an annealing-competent conformation.

Doetsch M, Fürtig B, Gstrein T, Stampfl S, Schroeder R - Nucleic Acids Res. (2011)

Bottom Line: In order to study the mechanism of protein-facilitated acceleration of annealing we selected a short peptide, HIV-1 Tat(44-61), which accelerates the reaction efficiently.Additionally, we found that Tat(44-61) drives the RNA annealing reaction via entropic rather than enthalpic terms.One-dimensional-NMR data suggest that the peptide changes the population distribution of possible RNA structures to favor an annealing-prone RNA conformation, thereby increasing the fraction of colliding RNA molecules that successfully anneal.

View Article: PubMed Central - PubMed

Affiliation: Max F Perutz Laboratories, Dr Bohrgasse 9/5, 1030 Vienna, Austria.

ABSTRACT
The annealing of nucleic acids to (partly) complementary RNA or DNA strands is involved in important cellular processes. A variety of proteins have been shown to accelerate RNA/RNA annealing but their mode of action is still mainly uncertain. In order to study the mechanism of protein-facilitated acceleration of annealing we selected a short peptide, HIV-1 Tat(44-61), which accelerates the reaction efficiently. The activity of the peptide is strongly regulated by mono- and divalent cations which hints at the importance of electrostatic interactions between RNA and peptide. Mutagenesis of the peptide illustrated the dominant role of positively charged amino acids in RNA annealing--both the overall charge of the molecule and a precise distribution of basic amino acids within the peptide are important. Additionally, we found that Tat(44-61) drives the RNA annealing reaction via entropic rather than enthalpic terms. One-dimensional-NMR data suggest that the peptide changes the population distribution of possible RNA structures to favor an annealing-prone RNA conformation, thereby increasing the fraction of colliding RNA molecules that successfully anneal.

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Aggregation and annealing acceleration by the Tat peptide do not correlate. (A) Aggregation assays with 21R+ ssRNA in the presence of 300 nM Tat(44–61), ∼0.1 µg/µl BSA or both proteins were carried out. While BSA did not aggregate the RNA, Tat(44–61) depleted >90% RNA from the solution. In the presence of BSA, however, Tat(44–61) had only a minor aggregation effect. (B) Interestingly, the same amount of BSA did not influence the RNA annealing activity of 300 nM of the peptide as determined using the FRET-based annealing assay. (C and D) RNA aggregation by 300 nM Tat(44–61) was assayed under different MgCl2 and NaCl concentrations that had been shown to inhibit the peptide’s annealing activity to a greater or lesser extent. However, no significant difference in measured aggregation was detectable.
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Figure 5: Aggregation and annealing acceleration by the Tat peptide do not correlate. (A) Aggregation assays with 21R+ ssRNA in the presence of 300 nM Tat(44–61), ∼0.1 µg/µl BSA or both proteins were carried out. While BSA did not aggregate the RNA, Tat(44–61) depleted >90% RNA from the solution. In the presence of BSA, however, Tat(44–61) had only a minor aggregation effect. (B) Interestingly, the same amount of BSA did not influence the RNA annealing activity of 300 nM of the peptide as determined using the FRET-based annealing assay. (C and D) RNA aggregation by 300 nM Tat(44–61) was assayed under different MgCl2 and NaCl concentrations that had been shown to inhibit the peptide’s annealing activity to a greater or lesser extent. However, no significant difference in measured aggregation was detectable.

Mentions: To address this hypothesis, we tested whether Tat(44–61) aggregates RNA with a simple centrifugation assay (46). We found the peptide to be positive in this assay using single-stranded 21R+ (Figure 5A) and double-stranded 21R RNA (data not shown). However, the addition of BSA to the reaction prevented Tat(44–61) caused depletion of RNA from the solution almost completely. Interestingly, BSA had no influence on the annealing activity of Tat(44–61) as assayed with the FRET-based annealing assay (Figure 5B). We therefore assume that the observed ‘aggregation’ was due to non-specific binding of the Tat peptide to test tube walls and that in contrast to Ncp7 no aggregates were formed. Further experiments supported this conclusion. MgCl2 and NaCl concentrations that inhibited annealing acceleration by the Tat peptide (Figure 2) did not affect the outcome of the aggregation assay (Figure 5C and D). In contrast, mono- and divalent ions have been shown to strongly influence Ncp7 induced nucleic acid aggregation (63). We could not detect a difference in RNA depletion for the three different peptide concentrations 100, 200 and 300 nM, which showed different magnitudes of annealing activity as well. Shorter centrifugation times resulted in less ‘aggregated’ RNA for all three concentrations without showing any significant discrimination between the three concentrations (Supplementary Figure S9).


The RNA annealing mechanism of the HIV-1 Tat peptide: conversion of the RNA into an annealing-competent conformation.

Doetsch M, Fürtig B, Gstrein T, Stampfl S, Schroeder R - Nucleic Acids Res. (2011)

Aggregation and annealing acceleration by the Tat peptide do not correlate. (A) Aggregation assays with 21R+ ssRNA in the presence of 300 nM Tat(44–61), ∼0.1 µg/µl BSA or both proteins were carried out. While BSA did not aggregate the RNA, Tat(44–61) depleted >90% RNA from the solution. In the presence of BSA, however, Tat(44–61) had only a minor aggregation effect. (B) Interestingly, the same amount of BSA did not influence the RNA annealing activity of 300 nM of the peptide as determined using the FRET-based annealing assay. (C and D) RNA aggregation by 300 nM Tat(44–61) was assayed under different MgCl2 and NaCl concentrations that had been shown to inhibit the peptide’s annealing activity to a greater or lesser extent. However, no significant difference in measured aggregation was detectable.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105384&req=5

Figure 5: Aggregation and annealing acceleration by the Tat peptide do not correlate. (A) Aggregation assays with 21R+ ssRNA in the presence of 300 nM Tat(44–61), ∼0.1 µg/µl BSA or both proteins were carried out. While BSA did not aggregate the RNA, Tat(44–61) depleted >90% RNA from the solution. In the presence of BSA, however, Tat(44–61) had only a minor aggregation effect. (B) Interestingly, the same amount of BSA did not influence the RNA annealing activity of 300 nM of the peptide as determined using the FRET-based annealing assay. (C and D) RNA aggregation by 300 nM Tat(44–61) was assayed under different MgCl2 and NaCl concentrations that had been shown to inhibit the peptide’s annealing activity to a greater or lesser extent. However, no significant difference in measured aggregation was detectable.
Mentions: To address this hypothesis, we tested whether Tat(44–61) aggregates RNA with a simple centrifugation assay (46). We found the peptide to be positive in this assay using single-stranded 21R+ (Figure 5A) and double-stranded 21R RNA (data not shown). However, the addition of BSA to the reaction prevented Tat(44–61) caused depletion of RNA from the solution almost completely. Interestingly, BSA had no influence on the annealing activity of Tat(44–61) as assayed with the FRET-based annealing assay (Figure 5B). We therefore assume that the observed ‘aggregation’ was due to non-specific binding of the Tat peptide to test tube walls and that in contrast to Ncp7 no aggregates were formed. Further experiments supported this conclusion. MgCl2 and NaCl concentrations that inhibited annealing acceleration by the Tat peptide (Figure 2) did not affect the outcome of the aggregation assay (Figure 5C and D). In contrast, mono- and divalent ions have been shown to strongly influence Ncp7 induced nucleic acid aggregation (63). We could not detect a difference in RNA depletion for the three different peptide concentrations 100, 200 and 300 nM, which showed different magnitudes of annealing activity as well. Shorter centrifugation times resulted in less ‘aggregated’ RNA for all three concentrations without showing any significant discrimination between the three concentrations (Supplementary Figure S9).

Bottom Line: In order to study the mechanism of protein-facilitated acceleration of annealing we selected a short peptide, HIV-1 Tat(44-61), which accelerates the reaction efficiently.Additionally, we found that Tat(44-61) drives the RNA annealing reaction via entropic rather than enthalpic terms.One-dimensional-NMR data suggest that the peptide changes the population distribution of possible RNA structures to favor an annealing-prone RNA conformation, thereby increasing the fraction of colliding RNA molecules that successfully anneal.

View Article: PubMed Central - PubMed

Affiliation: Max F Perutz Laboratories, Dr Bohrgasse 9/5, 1030 Vienna, Austria.

ABSTRACT
The annealing of nucleic acids to (partly) complementary RNA or DNA strands is involved in important cellular processes. A variety of proteins have been shown to accelerate RNA/RNA annealing but their mode of action is still mainly uncertain. In order to study the mechanism of protein-facilitated acceleration of annealing we selected a short peptide, HIV-1 Tat(44-61), which accelerates the reaction efficiently. The activity of the peptide is strongly regulated by mono- and divalent cations which hints at the importance of electrostatic interactions between RNA and peptide. Mutagenesis of the peptide illustrated the dominant role of positively charged amino acids in RNA annealing--both the overall charge of the molecule and a precise distribution of basic amino acids within the peptide are important. Additionally, we found that Tat(44-61) drives the RNA annealing reaction via entropic rather than enthalpic terms. One-dimensional-NMR data suggest that the peptide changes the population distribution of possible RNA structures to favor an annealing-prone RNA conformation, thereby increasing the fraction of colliding RNA molecules that successfully anneal.

Show MeSH
Related in: MedlinePlus