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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 peptide’s basic amino acids are crucial for the acceleration of annealing. (A) Amino acid sequence of Tat(44–61) with the positively charged amino acids in bold. (B) Different single amino acid mutants and a double and triple amino acid mutant of Tat(44–61) were tested in our FRET-based annealing assay with the 21R RNA pair at 30°C (using 300 nM peptide). Normalized FRET-values were fitted to the monophasic second-order reaction equation yielding the kobs for each peptide mutant. The obtained kobs were used to calculate the mutant activities relative to the WT as follows. In case the kobs(mutant) was higher than the kobs(WT), kobs(mutant) was divided by kobs(WT) and plotted as a positive value. In case of a decreased mutant kobs relative to the WT kobs, kobs(WT) was divided by kobs(mutant) and plotted as a negative value. This calculation method was used in order to emphasize specifically kobs decreases. Notably, the activity of all peptides in which a basic arginine or lysine was exchanged against alanine was reduced by a factor of 2.5–3 relative to the wild-type peptide. The double and triple mutant ‘K7A R13A’ and ‘K7A R9A R13A’ did not accelerate annealing detectably at the applied conditions. Two peptide mutants (I2T and Y4R) showed a higher annealing activity than Tat(44–61).
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Figure 3: The peptide’s basic amino acids are crucial for the acceleration of annealing. (A) Amino acid sequence of Tat(44–61) with the positively charged amino acids in bold. (B) Different single amino acid mutants and a double and triple amino acid mutant of Tat(44–61) were tested in our FRET-based annealing assay with the 21R RNA pair at 30°C (using 300 nM peptide). Normalized FRET-values were fitted to the monophasic second-order reaction equation yielding the kobs for each peptide mutant. The obtained kobs were used to calculate the mutant activities relative to the WT as follows. In case the kobs(mutant) was higher than the kobs(WT), kobs(mutant) was divided by kobs(WT) and plotted as a positive value. In case of a decreased mutant kobs relative to the WT kobs, kobs(WT) was divided by kobs(mutant) and plotted as a negative value. This calculation method was used in order to emphasize specifically kobs decreases. Notably, the activity of all peptides in which a basic arginine or lysine was exchanged against alanine was reduced by a factor of 2.5–3 relative to the wild-type peptide. The double and triple mutant ‘K7A R13A’ and ‘K7A R9A R13A’ did not accelerate annealing detectably at the applied conditions. Two peptide mutants (I2T and Y4R) showed a higher annealing activity than Tat(44–61).

Mentions: A set of peptides with single amino acid substitutions was therefore tested for their annealing activity in our FRET-based annealing assay (Figure 3, Supplementary Figure S7). Figure 3B shows the mutants’ kobs normalized to the wild-type kobs. Relative kobs values were confirmed for a few selected mutants using gel annealing assays (data not shown).Figure 3.


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 peptide’s basic amino acids are crucial for the acceleration of annealing. (A) Amino acid sequence of Tat(44–61) with the positively charged amino acids in bold. (B) Different single amino acid mutants and a double and triple amino acid mutant of Tat(44–61) were tested in our FRET-based annealing assay with the 21R RNA pair at 30°C (using 300 nM peptide). Normalized FRET-values were fitted to the monophasic second-order reaction equation yielding the kobs for each peptide mutant. The obtained kobs were used to calculate the mutant activities relative to the WT as follows. In case the kobs(mutant) was higher than the kobs(WT), kobs(mutant) was divided by kobs(WT) and plotted as a positive value. In case of a decreased mutant kobs relative to the WT kobs, kobs(WT) was divided by kobs(mutant) and plotted as a negative value. This calculation method was used in order to emphasize specifically kobs decreases. Notably, the activity of all peptides in which a basic arginine or lysine was exchanged against alanine was reduced by a factor of 2.5–3 relative to the wild-type peptide. The double and triple mutant ‘K7A R13A’ and ‘K7A R9A R13A’ did not accelerate annealing detectably at the applied conditions. Two peptide mutants (I2T and Y4R) showed a higher annealing activity than Tat(44–61).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: The peptide’s basic amino acids are crucial for the acceleration of annealing. (A) Amino acid sequence of Tat(44–61) with the positively charged amino acids in bold. (B) Different single amino acid mutants and a double and triple amino acid mutant of Tat(44–61) were tested in our FRET-based annealing assay with the 21R RNA pair at 30°C (using 300 nM peptide). Normalized FRET-values were fitted to the monophasic second-order reaction equation yielding the kobs for each peptide mutant. The obtained kobs were used to calculate the mutant activities relative to the WT as follows. In case the kobs(mutant) was higher than the kobs(WT), kobs(mutant) was divided by kobs(WT) and plotted as a positive value. In case of a decreased mutant kobs relative to the WT kobs, kobs(WT) was divided by kobs(mutant) and plotted as a negative value. This calculation method was used in order to emphasize specifically kobs decreases. Notably, the activity of all peptides in which a basic arginine or lysine was exchanged against alanine was reduced by a factor of 2.5–3 relative to the wild-type peptide. The double and triple mutant ‘K7A R13A’ and ‘K7A R9A R13A’ did not accelerate annealing detectably at the applied conditions. Two peptide mutants (I2T and Y4R) showed a higher annealing activity than Tat(44–61).
Mentions: A set of peptides with single amino acid substitutions was therefore tested for their annealing activity in our FRET-based annealing assay (Figure 3, Supplementary Figure S7). Figure 3B shows the mutants’ kobs normalized to the wild-type kobs. Relative kobs values were confirmed for a few selected mutants using gel annealing assays (data not shown).Figure 3.

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