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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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Mono- and divalent cations are detrimental to the peptide’s annealing activity. FRET-based annealing assays with 21R RNA were carried out in the absence or presence of 300 nM Tat(44–61), and at different MgCl2 and NaCl concentrations. The reaction constants (kobs) of the reaction in the presence of peptide were divided by the kobs of the ‘RNA only’ reaction, yielding kacc. Acceleration of annealing by the peptide was strongly impaired at MgCl2 concentrations above 2 mM and NaCl concentrations >60 mM.
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Figure 2: Mono- and divalent cations are detrimental to the peptide’s annealing activity. FRET-based annealing assays with 21R RNA were carried out in the absence or presence of 300 nM Tat(44–61), and at different MgCl2 and NaCl concentrations. The reaction constants (kobs) of the reaction in the presence of peptide were divided by the kobs of the ‘RNA only’ reaction, yielding kacc. Acceleration of annealing by the peptide was strongly impaired at MgCl2 concentrations above 2 mM and NaCl concentrations >60 mM.

Mentions: Tat(44–61) accelerated annealing with very similar kobs between pH 6.5 and 7.5 (Supplementary Figure S6). We thus measured acceleration of annealing at pH 7. We also tested the influence of mono- and divalent cations on the peptide’s activity (Figure 2). Consistent with the kinetic salt effect, the reaction constant of the ‘RNA only’ reaction increased with rising NaCl or MgCl2 concentration (47). In comparison to ‘no salt’ conditions 100 mM NaCl and 10 mM MgCl2 accelerated annealing 1.5- and 3-fold, respectively (data not shown). Tat(44–61) does not require any cations for its annealing activity: the peptide accelerated annealing ∼7- to 8-fold when no or only low amounts of ions were present. These kacc values [with kacc = kobs(peptide)/kobs(RNA only)] are in good agreement with the calculated acceleration of annealing measured with gel annealing assays (kacc = 6 and 9.5 at 10 or 20°C, respectively, as derived from the kobs values in Figure 6).Figure 2.


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)

Mono- and divalent cations are detrimental to the peptide’s annealing activity. FRET-based annealing assays with 21R RNA were carried out in the absence or presence of 300 nM Tat(44–61), and at different MgCl2 and NaCl concentrations. The reaction constants (kobs) of the reaction in the presence of peptide were divided by the kobs of the ‘RNA only’ reaction, yielding kacc. Acceleration of annealing by the peptide was strongly impaired at MgCl2 concentrations above 2 mM and NaCl concentrations >60 mM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Mono- and divalent cations are detrimental to the peptide’s annealing activity. FRET-based annealing assays with 21R RNA were carried out in the absence or presence of 300 nM Tat(44–61), and at different MgCl2 and NaCl concentrations. The reaction constants (kobs) of the reaction in the presence of peptide were divided by the kobs of the ‘RNA only’ reaction, yielding kacc. Acceleration of annealing by the peptide was strongly impaired at MgCl2 concentrations above 2 mM and NaCl concentrations >60 mM.
Mentions: Tat(44–61) accelerated annealing with very similar kobs between pH 6.5 and 7.5 (Supplementary Figure S6). We thus measured acceleration of annealing at pH 7. We also tested the influence of mono- and divalent cations on the peptide’s activity (Figure 2). Consistent with the kinetic salt effect, the reaction constant of the ‘RNA only’ reaction increased with rising NaCl or MgCl2 concentration (47). In comparison to ‘no salt’ conditions 100 mM NaCl and 10 mM MgCl2 accelerated annealing 1.5- and 3-fold, respectively (data not shown). Tat(44–61) does not require any cations for its annealing activity: the peptide accelerated annealing ∼7- to 8-fold when no or only low amounts of ions were present. These kacc values [with kacc = kobs(peptide)/kobs(RNA only)] are in good agreement with the calculated acceleration of annealing measured with gel annealing assays (kacc = 6 and 9.5 at 10 or 20°C, respectively, as derived from the kobs values in Figure 6).Figure 2.

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