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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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Tat(44–61) selects a specific RNA structure. 1H 1D NMR spectra of 21R+, a 21nt single stranded RNA, in the absence (black) or the presence (blue) of two equivalents of Tat(44–61) at different temperatures (indicated in gray at the right panel of the figure) were recorded. The chemical shifts of the aromatic protons did not overlap with the peptide chemical shifts and serve thus as an indicator for the peptide’s influence on the RNA conformation: the only visible peaks of the peptide in this spectral region are stemming from the aromatic protons of Y47 and are marked with asterisks. To follow changes in the spectrum upon addition of peptide gray dotted lines are added exemplarily, the symbol indicate if peaks are shifting upon addition of the peptide (less than and greater than symbol) or show no change (struck through circle).
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Figure 7: Tat(44–61) selects a specific RNA structure. 1H 1D NMR spectra of 21R+, a 21nt single stranded RNA, in the absence (black) or the presence (blue) of two equivalents of Tat(44–61) at different temperatures (indicated in gray at the right panel of the figure) were recorded. The chemical shifts of the aromatic protons did not overlap with the peptide chemical shifts and serve thus as an indicator for the peptide’s influence on the RNA conformation: the only visible peaks of the peptide in this spectral region are stemming from the aromatic protons of Y47 and are marked with asterisks. To follow changes in the spectrum upon addition of peptide gray dotted lines are added exemplarily, the symbol indicate if peaks are shifting upon addition of the peptide (less than and greater than symbol) or show no change (struck through circle).

Mentions: In order to learn more about the structural influence of Tat(44–61) on its RNA substrate we recorded 1D 1H spectra of the 21R + RNA in the absence or presence of different amounts of Tat peptide and at different temperatures (Figure 7).Figure 7.


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)

Tat(44–61) selects a specific RNA structure. 1H 1D NMR spectra of 21R+, a 21nt single stranded RNA, in the absence (black) or the presence (blue) of two equivalents of Tat(44–61) at different temperatures (indicated in gray at the right panel of the figure) were recorded. The chemical shifts of the aromatic protons did not overlap with the peptide chemical shifts and serve thus as an indicator for the peptide’s influence on the RNA conformation: the only visible peaks of the peptide in this spectral region are stemming from the aromatic protons of Y47 and are marked with asterisks. To follow changes in the spectrum upon addition of peptide gray dotted lines are added exemplarily, the symbol indicate if peaks are shifting upon addition of the peptide (less than and greater than symbol) or show no change (struck through circle).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 7: Tat(44–61) selects a specific RNA structure. 1H 1D NMR spectra of 21R+, a 21nt single stranded RNA, in the absence (black) or the presence (blue) of two equivalents of Tat(44–61) at different temperatures (indicated in gray at the right panel of the figure) were recorded. The chemical shifts of the aromatic protons did not overlap with the peptide chemical shifts and serve thus as an indicator for the peptide’s influence on the RNA conformation: the only visible peaks of the peptide in this spectral region are stemming from the aromatic protons of Y47 and are marked with asterisks. To follow changes in the spectrum upon addition of peptide gray dotted lines are added exemplarily, the symbol indicate if peaks are shifting upon addition of the peptide (less than and greater than symbol) or show no change (struck through circle).
Mentions: In order to learn more about the structural influence of Tat(44–61) on its RNA substrate we recorded 1D 1H spectra of the 21R + RNA in the absence or presence of different amounts of Tat peptide and at different temperatures (Figure 7).Figure 7.

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