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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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The spatial arrangement of basic amino acids within the peptide is important for annealing activity. (A) Primary sequences of scrambled peptides with the same amino acid composition as the WT Tat(44–61) peptide. (B) The FRET-based annealing assay using 21R RNA was carried out at 30°C. The obtained reaction constant kobs of each peptide mutant was normalized to the WT kobs, yielding the ‘fold activity relative to the WT’. Interestingly, all scrambled peptides containing the same amount of basic amino acids as Tat(44–61) were less active than the WT.
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Figure 4: The spatial arrangement of basic amino acids within the peptide is important for annealing activity. (A) Primary sequences of scrambled peptides with the same amino acid composition as the WT Tat(44–61) peptide. (B) The FRET-based annealing assay using 21R RNA was carried out at 30°C. The obtained reaction constant kobs of each peptide mutant was normalized to the WT kobs, yielding the ‘fold activity relative to the WT’. Interestingly, all scrambled peptides containing the same amount of basic amino acids as Tat(44–61) were less active than the WT.

Mentions: The results discussed in the previous section imply that the most important factor that confers annealing activity is the overall charge of the peptide. This could mean that the peptide acts as an octovalent ion. To assess the question as to whether a specific spatial arrangement of basic amino acids within the peptide is important we scrambled the peptide in three different ways (Figure 4A): (i) in the mutant scr1 the eight basic amino acids are distributed evenly over the peptide; (ii) scr3 contains two stretches of basic residues, as does the WT peptide, however these are separated by a larger amount of uncharged amino acids; (iii) scr2 contains only one basic stretch while the other basic residues are more distributed.Figure 4.


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)

The spatial arrangement of basic amino acids within the peptide is important for annealing activity. (A) Primary sequences of scrambled peptides with the same amino acid composition as the WT Tat(44–61) peptide. (B) The FRET-based annealing assay using 21R RNA was carried out at 30°C. The obtained reaction constant kobs of each peptide mutant was normalized to the WT kobs, yielding the ‘fold activity relative to the WT’. Interestingly, all scrambled peptides containing the same amount of basic amino acids as Tat(44–61) were less active than the WT.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: The spatial arrangement of basic amino acids within the peptide is important for annealing activity. (A) Primary sequences of scrambled peptides with the same amino acid composition as the WT Tat(44–61) peptide. (B) The FRET-based annealing assay using 21R RNA was carried out at 30°C. The obtained reaction constant kobs of each peptide mutant was normalized to the WT kobs, yielding the ‘fold activity relative to the WT’. Interestingly, all scrambled peptides containing the same amount of basic amino acids as Tat(44–61) were less active than the WT.
Mentions: The results discussed in the previous section imply that the most important factor that confers annealing activity is the overall charge of the peptide. This could mean that the peptide acts as an octovalent ion. To assess the question as to whether a specific spatial arrangement of basic amino acids within the peptide is important we scrambled the peptide in three different ways (Figure 4A): (i) in the mutant scr1 the eight basic amino acids are distributed evenly over the peptide; (ii) scr3 contains two stretches of basic residues, as does the WT peptide, however these are separated by a larger amount of uncharged amino acids; (iii) scr2 contains only one basic stretch while the other basic residues are more distributed.Figure 4.

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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