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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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Schematic representation of peptide-induced RNA structural changes. (A) RNA only, (B) RNA in the presence of Tat(44–61) with its N-terminus in blue and its C-terminus in red. At low temperature, the interaction of the peptide with the RNA induces changes in the conformational equilibrium in a way that not only a single helical conformation is present but the helical-conformation is locally disrupted or partially melted. At intermediate temperatures (close to the ‘melting point’) the RNA is anyhow in a conformational equilibrium between helical and partially melted conformations. The addition of peptide to this pool of conformers results in a partial stabilization of the partially melted RNA conformations, which are probably the ones where RNA duplex formation initiates. At high temperatures, Tat(44–61) will stabilize not the completely randomized unfolded form of the RNA, but intermediates that are prone to interaction with a complementary strand of RNA.
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Figure 8: Schematic representation of peptide-induced RNA structural changes. (A) RNA only, (B) RNA in the presence of Tat(44–61) with its N-terminus in blue and its C-terminus in red. At low temperature, the interaction of the peptide with the RNA induces changes in the conformational equilibrium in a way that not only a single helical conformation is present but the helical-conformation is locally disrupted or partially melted. At intermediate temperatures (close to the ‘melting point’) the RNA is anyhow in a conformational equilibrium between helical and partially melted conformations. The addition of peptide to this pool of conformers results in a partial stabilization of the partially melted RNA conformations, which are probably the ones where RNA duplex formation initiates. At high temperatures, Tat(44–61) will stabilize not the completely randomized unfolded form of the RNA, but intermediates that are prone to interaction with a complementary strand of RNA.

Mentions: We conclude that at low temperatures a distinct, probably helical conformation is adopted by the single-stranded 21R+ RNA (Figure 8A). When the temperature is raised, this conformation starts to melt. Around 20–25°C the molecule is no longer best described by a single static conformation but by an ensemble of interconverting partially melted ‘helices’. The temperature increase to 47°C converts the 21R+ RNA into similar, unstructured (and most probably unstacked) conformations that give rise to the low dispersion but narrow line-shape signature of the peaks. This observation is in accordance with CD-spectroscopic melting experiments (Supplementary Figure S12). When the CD-temperature row is analyzed for a two-state unfolding process, the melting temperature is determined to be Tm = 38°C meaning that at this particular temperature the RNA is in an equilibrium of 50% unfolded and folded conformations.Figure 8.


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)

Schematic representation of peptide-induced RNA structural changes. (A) RNA only, (B) RNA in the presence of Tat(44–61) with its N-terminus in blue and its C-terminus in red. At low temperature, the interaction of the peptide with the RNA induces changes in the conformational equilibrium in a way that not only a single helical conformation is present but the helical-conformation is locally disrupted or partially melted. At intermediate temperatures (close to the ‘melting point’) the RNA is anyhow in a conformational equilibrium between helical and partially melted conformations. The addition of peptide to this pool of conformers results in a partial stabilization of the partially melted RNA conformations, which are probably the ones where RNA duplex formation initiates. At high temperatures, Tat(44–61) will stabilize not the completely randomized unfolded form of the RNA, but intermediates that are prone to interaction with a complementary strand of RNA.
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Related In: Results  -  Collection

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Show All Figures
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Figure 8: Schematic representation of peptide-induced RNA structural changes. (A) RNA only, (B) RNA in the presence of Tat(44–61) with its N-terminus in blue and its C-terminus in red. At low temperature, the interaction of the peptide with the RNA induces changes in the conformational equilibrium in a way that not only a single helical conformation is present but the helical-conformation is locally disrupted or partially melted. At intermediate temperatures (close to the ‘melting point’) the RNA is anyhow in a conformational equilibrium between helical and partially melted conformations. The addition of peptide to this pool of conformers results in a partial stabilization of the partially melted RNA conformations, which are probably the ones where RNA duplex formation initiates. At high temperatures, Tat(44–61) will stabilize not the completely randomized unfolded form of the RNA, but intermediates that are prone to interaction with a complementary strand of RNA.
Mentions: We conclude that at low temperatures a distinct, probably helical conformation is adopted by the single-stranded 21R+ RNA (Figure 8A). When the temperature is raised, this conformation starts to melt. Around 20–25°C the molecule is no longer best described by a single static conformation but by an ensemble of interconverting partially melted ‘helices’. The temperature increase to 47°C converts the 21R+ RNA into similar, unstructured (and most probably unstacked) conformations that give rise to the low dispersion but narrow line-shape signature of the peaks. This observation is in accordance with CD-spectroscopic melting experiments (Supplementary Figure S12). When the CD-temperature row is analyzed for a two-state unfolding process, the melting temperature is determined to be Tm = 38°C meaning that at this particular temperature the RNA is in an equilibrium of 50% unfolded and folded conformations.Figure 8.

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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