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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) accelerates annealing of two complementary RNAs but does not promote strand displacement. (A) Scheme of the FRET-based combined annealing and strand displacement (SD) assay according to Rajkowitsch and Schroeder (44). Two short complementary RNAs, labeled with a Cy3- (donor) and a Cy5-dye (acceptor), respectively, are annealed in a microplate (phase I). The close proximity of the two dyes results in a fluorescence resonance energy transfer (FRET) when the donor dye is excited. Both fluorescent emission signals are measured at 1 s intervals using a microplate reader, and the FRET index is calculated as the ratio of the FRET to the Cy3-signal. Strand displacement is detected in phase II of the assay which is started through the addition of an unlabeled competitor RNA and results in a decrease of the FRET signal. (B) The assay was conducted with 21R RNA in the absence or presence of 1 µM Tat peptide and at 30°C. E. coli StpA (1 µM) was used as a positive control. For clarity, the FRET index was normalized to 1 (phase I and II) and 0 (only phase I).
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Figure 1: Tat(44–61) accelerates annealing of two complementary RNAs but does not promote strand displacement. (A) Scheme of the FRET-based combined annealing and strand displacement (SD) assay according to Rajkowitsch and Schroeder (44). Two short complementary RNAs, labeled with a Cy3- (donor) and a Cy5-dye (acceptor), respectively, are annealed in a microplate (phase I). The close proximity of the two dyes results in a fluorescence resonance energy transfer (FRET) when the donor dye is excited. Both fluorescent emission signals are measured at 1 s intervals using a microplate reader, and the FRET index is calculated as the ratio of the FRET to the Cy3-signal. Strand displacement is detected in phase II of the assay which is started through the addition of an unlabeled competitor RNA and results in a decrease of the FRET signal. (B) The assay was conducted with 21R RNA in the absence or presence of 1 µM Tat peptide and at 30°C. E. coli StpA (1 µM) was used as a positive control. For clarity, the FRET index was normalized to 1 (phase I and II) and 0 (only phase I).

Mentions: In order to confirm the described activities we used Tat(44–61) in a two-phase FRET-based assay (Figure 1A) that has previously been described and used for several potential RNA chaperone proteins (14,44). As anticipated, Tat(44–61) considerably accelerated annealing of the 21R substrate (Supplementary Table S1), a 21 base-pair long, blunt-ended, artificial RNA duplex [Tm = 72.4°C, calculated using the DINAMelt server (52)] (Figure 1B, phase I). The peptide was however not active in the second (strand displacement) phase of the assay while the positive control StpA efficiently catalyzed strand displacement. Remarkably, Tat(44–61) also accelerated annealing of the DNA substrate 21D while not catalyzing strand displacement by a competitor DNA (Supplementary Figure S1). To exclude the possibility of the 21R double-stranded substrate being too stable for peptide mediated unfolding we used the jm1 substrate which has a lower GC content (Tm = 49.3°C), with the same result (Supplementary Table S1 and Figure S2C). Some proteins are known to need a single-stranded or helical binding platform to perform double-strand unwinding (8). To test this possibility for Tat(44–61) we used a 46 nt RNA, which forms a short hairpin at the 3′-end, together with a partly complementary 21-mer in the combined FRET-based assay (jm1heli substrate). Annealing of this substrate was accelerated by the peptide, whereas no strand displacement was detected (Supplementary Figure S2D). Other variations of the assay included using different temperatures (30 and 37°C), testing several peptide to nucleotide ratios (10 nM to 1 mM peptide per 10 nM RNA), changing the concentrations of MgCl2 and NaCl, using exactly the same buffer composition as described by ref. (42) (buffer A) as well as testing peptide buffers with different ZnCl2 concentrations. While we could always detect an acceleration of annealing, the peptide did not catalyze strand displacement under any of the applied conditions (Supplementary Figure S2A, B, E and F).Figure 1.


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) accelerates annealing of two complementary RNAs but does not promote strand displacement. (A) Scheme of the FRET-based combined annealing and strand displacement (SD) assay according to Rajkowitsch and Schroeder (44). Two short complementary RNAs, labeled with a Cy3- (donor) and a Cy5-dye (acceptor), respectively, are annealed in a microplate (phase I). The close proximity of the two dyes results in a fluorescence resonance energy transfer (FRET) when the donor dye is excited. Both fluorescent emission signals are measured at 1 s intervals using a microplate reader, and the FRET index is calculated as the ratio of the FRET to the Cy3-signal. Strand displacement is detected in phase II of the assay which is started through the addition of an unlabeled competitor RNA and results in a decrease of the FRET signal. (B) The assay was conducted with 21R RNA in the absence or presence of 1 µM Tat peptide and at 30°C. E. coli StpA (1 µM) was used as a positive control. For clarity, the FRET index was normalized to 1 (phase I and II) and 0 (only phase I).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Tat(44–61) accelerates annealing of two complementary RNAs but does not promote strand displacement. (A) Scheme of the FRET-based combined annealing and strand displacement (SD) assay according to Rajkowitsch and Schroeder (44). Two short complementary RNAs, labeled with a Cy3- (donor) and a Cy5-dye (acceptor), respectively, are annealed in a microplate (phase I). The close proximity of the two dyes results in a fluorescence resonance energy transfer (FRET) when the donor dye is excited. Both fluorescent emission signals are measured at 1 s intervals using a microplate reader, and the FRET index is calculated as the ratio of the FRET to the Cy3-signal. Strand displacement is detected in phase II of the assay which is started through the addition of an unlabeled competitor RNA and results in a decrease of the FRET signal. (B) The assay was conducted with 21R RNA in the absence or presence of 1 µM Tat peptide and at 30°C. E. coli StpA (1 µM) was used as a positive control. For clarity, the FRET index was normalized to 1 (phase I and II) and 0 (only phase I).
Mentions: In order to confirm the described activities we used Tat(44–61) in a two-phase FRET-based assay (Figure 1A) that has previously been described and used for several potential RNA chaperone proteins (14,44). As anticipated, Tat(44–61) considerably accelerated annealing of the 21R substrate (Supplementary Table S1), a 21 base-pair long, blunt-ended, artificial RNA duplex [Tm = 72.4°C, calculated using the DINAMelt server (52)] (Figure 1B, phase I). The peptide was however not active in the second (strand displacement) phase of the assay while the positive control StpA efficiently catalyzed strand displacement. Remarkably, Tat(44–61) also accelerated annealing of the DNA substrate 21D while not catalyzing strand displacement by a competitor DNA (Supplementary Figure S1). To exclude the possibility of the 21R double-stranded substrate being too stable for peptide mediated unfolding we used the jm1 substrate which has a lower GC content (Tm = 49.3°C), with the same result (Supplementary Table S1 and Figure S2C). Some proteins are known to need a single-stranded or helical binding platform to perform double-strand unwinding (8). To test this possibility for Tat(44–61) we used a 46 nt RNA, which forms a short hairpin at the 3′-end, together with a partly complementary 21-mer in the combined FRET-based assay (jm1heli substrate). Annealing of this substrate was accelerated by the peptide, whereas no strand displacement was detected (Supplementary Figure S2D). Other variations of the assay included using different temperatures (30 and 37°C), testing several peptide to nucleotide ratios (10 nM to 1 mM peptide per 10 nM RNA), changing the concentrations of MgCl2 and NaCl, using exactly the same buffer composition as described by ref. (42) (buffer A) as well as testing peptide buffers with different ZnCl2 concentrations. While we could always detect an acceleration of annealing, the peptide did not catalyze strand displacement under any of the applied conditions (Supplementary Figure S2A, B, E and F).Figure 1.

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