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The Interaction between tRNA(Lys) 3 and the primer activation signal deciphered by NMR spectroscopy.

Sleiman D, Barraud P, Brachet F, Tisne C - PLoS ONE (2013)

Bottom Line: Despite the fact that the result of this rearrangement is thermodynamically more stable, there is a high-energy barrier that requires the chaperoning activity of the viral nucleocapsid protein.Our NMR study provides molecular evidence of the existence of this interaction and highlights the role of the nucleocapsid protein in promoting this additional RNA-RNA annealing.This study presents the first direct observation at a single base-pair resolution of the PAS/anti-PAS association, which has been proposed to be involved in the chronological regulation of the reverse transcription.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Cristallographie et RMN biologiques, CNRS, Université Paris Descartes, Paris Sorbonne Cité, Paris, France.

ABSTRACT
The initiation of reverse transcription of the human immunodeficiency virus type 1 (HIV-1) requires the opening of the three-dimensional structure of the primer tRNA(Lys) 3 for its annealing to the viral RNA at the primer binding site (PBS). Despite the fact that the result of this rearrangement is thermodynamically more stable, there is a high-energy barrier that requires the chaperoning activity of the viral nucleocapsid protein. In addition to the nucleotide complementarity to the PBS, several regions of tRNA(Lys) 3 have been described as interacting with the viral genomic RNA. Among these sequences, a sequence of the viral genome called PAS for "primer activation signal" was proposed to interact with the T-arm of tRNA(Lys) 3, this interaction stimulating the initiation of reverse transcription. In this report, we investigate the formation of this additional interaction with NMR spectroscopy, using a simple system composed of the primer tRNA(Lys) 3, the 18 nucleotides of the PBS, the PAS (8 nucleotides) encompassed or not in a hairpin structure, and the nucleocapsid protein. Our NMR study provides molecular evidence of the existence of this interaction and highlights the role of the nucleocapsid protein in promoting this additional RNA-RNA annealing. This study presents the first direct observation at a single base-pair resolution of the PAS/anti-PAS association, which has been proposed to be involved in the chronological regulation of the reverse transcription.

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Related in: MedlinePlus

Test of annealing of PASds to tRNALys3 in presence of the PBS using the heat-annealed procedure.A) Schematic drawing of the annealing procedure, B) Superimposition of two TROSY experiments recorded at 15°C showing the imino groups of tRNALys3 alone (in black, reference spectrum) and after the heat-annealed procedure with 1 equivalent of PBS and PASds (red spectrum).
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pone-0064700-g006: Test of annealing of PASds to tRNALys3 in presence of the PBS using the heat-annealed procedure.A) Schematic drawing of the annealing procedure, B) Superimposition of two TROSY experiments recorded at 15°C showing the imino groups of tRNALys3 alone (in black, reference spectrum) and after the heat-annealed procedure with 1 equivalent of PBS and PASds (red spectrum).

Mentions: From a thermodynamic point of view, the annealing of tRNALys3 to the PBS is strongly favoured (Figure 5, ΔG  = −36.2 kcal/mol and Tmc  = 92°C). In addition, the annealing of tRNALys3 to the PBS will partially open the tRNA structure and thus increase the accessibility to the anti-PAS sequence. Therefore, in order to investigate whether the PBS could influence the annealing of the PAS sequence with tRNALys3, we decided to conduct the heat-annealed procedure in the presence of the PBS. Figure 6 shows the NMR footprint experiment resulting from this procedure. The assignment of tRNALys3/PBS imino groups was previously published [7]. The imino groups of tRNALys3 (black spectrum – Figure 6) have disappeared to give rise to imino groups of tRNALys3 involved in base-pairing with the PBS (red spectrum – Figure 6). A NOESY experiment (data not shown) substantiates that the PASds is still free as its imino groups are still observable at the chemical shifts of the imino groups of the free PASds like in Figure 4. In conclusion, when we mixed 15N-tRNALys3 to the PBS and to the PASds at the same time, the heat-annealed complex is composed of the PBS bound to tRNALys3 and the PAS remains free in solution (Figure 6). This complex is stable and does not change with time.


The Interaction between tRNA(Lys) 3 and the primer activation signal deciphered by NMR spectroscopy.

Sleiman D, Barraud P, Brachet F, Tisne C - PLoS ONE (2013)

Test of annealing of PASds to tRNALys3 in presence of the PBS using the heat-annealed procedure.A) Schematic drawing of the annealing procedure, B) Superimposition of two TROSY experiments recorded at 15°C showing the imino groups of tRNALys3 alone (in black, reference spectrum) and after the heat-annealed procedure with 1 equivalent of PBS and PASds (red spectrum).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675109&req=5

pone-0064700-g006: Test of annealing of PASds to tRNALys3 in presence of the PBS using the heat-annealed procedure.A) Schematic drawing of the annealing procedure, B) Superimposition of two TROSY experiments recorded at 15°C showing the imino groups of tRNALys3 alone (in black, reference spectrum) and after the heat-annealed procedure with 1 equivalent of PBS and PASds (red spectrum).
Mentions: From a thermodynamic point of view, the annealing of tRNALys3 to the PBS is strongly favoured (Figure 5, ΔG  = −36.2 kcal/mol and Tmc  = 92°C). In addition, the annealing of tRNALys3 to the PBS will partially open the tRNA structure and thus increase the accessibility to the anti-PAS sequence. Therefore, in order to investigate whether the PBS could influence the annealing of the PAS sequence with tRNALys3, we decided to conduct the heat-annealed procedure in the presence of the PBS. Figure 6 shows the NMR footprint experiment resulting from this procedure. The assignment of tRNALys3/PBS imino groups was previously published [7]. The imino groups of tRNALys3 (black spectrum – Figure 6) have disappeared to give rise to imino groups of tRNALys3 involved in base-pairing with the PBS (red spectrum – Figure 6). A NOESY experiment (data not shown) substantiates that the PASds is still free as its imino groups are still observable at the chemical shifts of the imino groups of the free PASds like in Figure 4. In conclusion, when we mixed 15N-tRNALys3 to the PBS and to the PASds at the same time, the heat-annealed complex is composed of the PBS bound to tRNALys3 and the PAS remains free in solution (Figure 6). This complex is stable and does not change with time.

Bottom Line: Despite the fact that the result of this rearrangement is thermodynamically more stable, there is a high-energy barrier that requires the chaperoning activity of the viral nucleocapsid protein.Our NMR study provides molecular evidence of the existence of this interaction and highlights the role of the nucleocapsid protein in promoting this additional RNA-RNA annealing.This study presents the first direct observation at a single base-pair resolution of the PAS/anti-PAS association, which has been proposed to be involved in the chronological regulation of the reverse transcription.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Cristallographie et RMN biologiques, CNRS, Université Paris Descartes, Paris Sorbonne Cité, Paris, France.

ABSTRACT
The initiation of reverse transcription of the human immunodeficiency virus type 1 (HIV-1) requires the opening of the three-dimensional structure of the primer tRNA(Lys) 3 for its annealing to the viral RNA at the primer binding site (PBS). Despite the fact that the result of this rearrangement is thermodynamically more stable, there is a high-energy barrier that requires the chaperoning activity of the viral nucleocapsid protein. In addition to the nucleotide complementarity to the PBS, several regions of tRNA(Lys) 3 have been described as interacting with the viral genomic RNA. Among these sequences, a sequence of the viral genome called PAS for "primer activation signal" was proposed to interact with the T-arm of tRNA(Lys) 3, this interaction stimulating the initiation of reverse transcription. In this report, we investigate the formation of this additional interaction with NMR spectroscopy, using a simple system composed of the primer tRNA(Lys) 3, the 18 nucleotides of the PBS, the PAS (8 nucleotides) encompassed or not in a hairpin structure, and the nucleocapsid protein. Our NMR study provides molecular evidence of the existence of this interaction and highlights the role of the nucleocapsid protein in promoting this additional RNA-RNA annealing. This study presents the first direct observation at a single base-pair resolution of the PAS/anti-PAS association, which has been proposed to be involved in the chronological regulation of the reverse transcription.

Show MeSH
Related in: MedlinePlus