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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) increases the transition state entropy of the annealing reaction. (A) RNA gel annealing assays were conducted in the absence or presence of protein and at different temperatures. To start the reaction a Cy3-labeled RNA strand was added to the Cy5-labeled complementary RNA. Aliquots were drawn after certain time points and the reaction was stopped with a 60× excess of non-labeled competitor strand. (B) The samples were applied directly onto a running native PAGE. Since the Cy5-dyes add ‘size’ to the RNAs the heteroduplex of two labeled strands (**ds) runs higher than the complex of the Cy3- and the unlabeled strands (*ds). The Cy5- and Cy3-fluorescent signals were scanned and the Cy5-signals from single and double strands were quantified. (C) Plotting the ratio of double-stranded RNA to total amount of RNA for each time point of a reaction yielded annealing curves which were fitted with a monophasic second-order reaction equation with equimolar initial reaction concentration. With rising temperature the kobs of the annealing reaction in the presence or absence of 300 nM Tat(44–61) increased. The rate constants k, normalized to the RNA concentration, were as follows: Tat(44–61) 5°C 9.0 × 105 M−1s−1 (filled inverted triangle), 10°C 14.5 × 105 M−1s−1 (filled diamond), 15°C 29.0 × 105 M−1 s−1 (filled circle), 20°C 40.0 × 105 M−1s−1 (filled square); RNA only 10°C 2.4 × 105 M−1s−1 (open triangle), 20°C 4.2 × 105 M−1s−1 (open diamond), 30°C 7.3 × 105 M−1s−1 (open circle), 37°C 10.6 × 105 M−1s−1 (open square). Means of curves and rate constants k were calculated from three to four independent experiments. (D) The natural logarithms of the normalized reaction constants k were plotted against the inverse temperature demonstrating the linear correlation between the two values in the measured temperature range (Arrhenius plot).
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Figure 6: Tat(44–61) increases the transition state entropy of the annealing reaction. (A) RNA gel annealing assays were conducted in the absence or presence of protein and at different temperatures. To start the reaction a Cy3-labeled RNA strand was added to the Cy5-labeled complementary RNA. Aliquots were drawn after certain time points and the reaction was stopped with a 60× excess of non-labeled competitor strand. (B) The samples were applied directly onto a running native PAGE. Since the Cy5-dyes add ‘size’ to the RNAs the heteroduplex of two labeled strands (**ds) runs higher than the complex of the Cy3- and the unlabeled strands (*ds). The Cy5- and Cy3-fluorescent signals were scanned and the Cy5-signals from single and double strands were quantified. (C) Plotting the ratio of double-stranded RNA to total amount of RNA for each time point of a reaction yielded annealing curves which were fitted with a monophasic second-order reaction equation with equimolar initial reaction concentration. With rising temperature the kobs of the annealing reaction in the presence or absence of 300 nM Tat(44–61) increased. The rate constants k, normalized to the RNA concentration, were as follows: Tat(44–61) 5°C 9.0 × 105 M−1s−1 (filled inverted triangle), 10°C 14.5 × 105 M−1s−1 (filled diamond), 15°C 29.0 × 105 M−1 s−1 (filled circle), 20°C 40.0 × 105 M−1s−1 (filled square); RNA only 10°C 2.4 × 105 M−1s−1 (open triangle), 20°C 4.2 × 105 M−1s−1 (open diamond), 30°C 7.3 × 105 M−1s−1 (open circle), 37°C 10.6 × 105 M−1s−1 (open square). Means of curves and rate constants k were calculated from three to four independent experiments. (D) The natural logarithms of the normalized reaction constants k were plotted against the inverse temperature demonstrating the linear correlation between the two values in the measured temperature range (Arrhenius plot).

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)

Tat(44–61) increases the transition state entropy of the annealing reaction. (A) RNA gel annealing assays were conducted in the absence or presence of protein and at different temperatures. To start the reaction a Cy3-labeled RNA strand was added to the Cy5-labeled complementary RNA. Aliquots were drawn after certain time points and the reaction was stopped with a 60× excess of non-labeled competitor strand. (B) The samples were applied directly onto a running native PAGE. Since the Cy5-dyes add ‘size’ to the RNAs the heteroduplex of two labeled strands (**ds) runs higher than the complex of the Cy3- and the unlabeled strands (*ds). The Cy5- and Cy3-fluorescent signals were scanned and the Cy5-signals from single and double strands were quantified. (C) Plotting the ratio of double-stranded RNA to total amount of RNA for each time point of a reaction yielded annealing curves which were fitted with a monophasic second-order reaction equation with equimolar initial reaction concentration. With rising temperature the kobs of the annealing reaction in the presence or absence of 300 nM Tat(44–61) increased. The rate constants k, normalized to the RNA concentration, were as follows: Tat(44–61) 5°C 9.0 × 105 M−1s−1 (filled inverted triangle), 10°C 14.5 × 105 M−1s−1 (filled diamond), 15°C 29.0 × 105 M−1 s−1 (filled circle), 20°C 40.0 × 105 M−1s−1 (filled square); RNA only 10°C 2.4 × 105 M−1s−1 (open triangle), 20°C 4.2 × 105 M−1s−1 (open diamond), 30°C 7.3 × 105 M−1s−1 (open circle), 37°C 10.6 × 105 M−1s−1 (open square). Means of curves and rate constants k were calculated from three to four independent experiments. (D) The natural logarithms of the normalized reaction constants k were plotted against the inverse temperature demonstrating the linear correlation between the two values in the measured temperature range (Arrhenius plot).
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Figure 6: Tat(44–61) increases the transition state entropy of the annealing reaction. (A) RNA gel annealing assays were conducted in the absence or presence of protein and at different temperatures. To start the reaction a Cy3-labeled RNA strand was added to the Cy5-labeled complementary RNA. Aliquots were drawn after certain time points and the reaction was stopped with a 60× excess of non-labeled competitor strand. (B) The samples were applied directly onto a running native PAGE. Since the Cy5-dyes add ‘size’ to the RNAs the heteroduplex of two labeled strands (**ds) runs higher than the complex of the Cy3- and the unlabeled strands (*ds). The Cy5- and Cy3-fluorescent signals were scanned and the Cy5-signals from single and double strands were quantified. (C) Plotting the ratio of double-stranded RNA to total amount of RNA for each time point of a reaction yielded annealing curves which were fitted with a monophasic second-order reaction equation with equimolar initial reaction concentration. With rising temperature the kobs of the annealing reaction in the presence or absence of 300 nM Tat(44–61) increased. The rate constants k, normalized to the RNA concentration, were as follows: Tat(44–61) 5°C 9.0 × 105 M−1s−1 (filled inverted triangle), 10°C 14.5 × 105 M−1s−1 (filled diamond), 15°C 29.0 × 105 M−1 s−1 (filled circle), 20°C 40.0 × 105 M−1s−1 (filled square); RNA only 10°C 2.4 × 105 M−1s−1 (open triangle), 20°C 4.2 × 105 M−1s−1 (open diamond), 30°C 7.3 × 105 M−1s−1 (open circle), 37°C 10.6 × 105 M−1s−1 (open square). Means of curves and rate constants k were calculated from three to four independent experiments. (D) The natural logarithms of the normalized reaction constants k were plotted against the inverse temperature demonstrating the linear correlation between the two values in the measured temperature range (Arrhenius plot).
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