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Core flexibility of a truncated metazoan mitochondrial tRNA.

Frazer-Abel AA, Hagerman PJ - Nucleic Acids Res. (2008)

Bottom Line: Thus, the absence of canonical TpsiC-D interactions likely results in greater dispersion of anticodon-acceptor interstem angle than for canonical tRNAs.To test this hypothesis, we have assessed the dispersion of the anticodon-acceptor angle for bovine mtRNA(Ser)(AGY), which lacks the canonical D arm and is thus incapable of forming stabilizing interarm interactions.These results suggest that increased flexibility, in addition to a more open interstem angle, would allow both noncanonical and canonical mtRNAs to utilize the same protein synthetic apparatus.

View Article: PubMed Central - PubMed

Affiliation: National Jewish Health, Denver, CO 80206, USA.

ABSTRACT
Secondary and tertiary structures of tRNAs are remarkably preserved from bacteria to humans, the notable exception being the mitochondrial (m) tRNAs of metazoans, which often deviate substantially from the canonical cloverleaf (secondary) or 'L'-shaped (tertiary) structure. Many metazoan mtRNAs lack either the TpsiC (T) or dihydrouridine (D) loops of the canonical cloverleaf, which are known to confer structural rigidity to the folded structure. Thus, the absence of canonical TpsiC-D interactions likely results in greater dispersion of anticodon-acceptor interstem angle than for canonical tRNAs. To test this hypothesis, we have assessed the dispersion of the anticodon-acceptor angle for bovine mtRNA(Ser)(AGY), which lacks the canonical D arm and is thus incapable of forming stabilizing interarm interactions. Using the method of transient electric birefringence (TEB), and by changing the helical torsion angle between a core mtRNA bend and a second bend of known angle/rigidity, we have demonstrated that the core of mtRNA(Ser)(AGY) has substantially greater flexibility than its well-characterized canonical counterpart, yeast cytoplasmic tRNA(Phe). These results suggest that increased flexibility, in addition to a more open interstem angle, would allow both noncanonical and canonical mtRNAs to utilize the same protein synthetic apparatus.

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Related in: MedlinePlus

Dependence of the linear length (bp) versus τ-value. Interpolations used for single-bend temperature data presented in panels A and B: (black circle) 0 mM MgCl2, 3.8°C; (white circle) 0 mM MgCl2, 20.8°C; (grey circle) 0 mM MgCl2, 37.4°C; (black square) 2 mM MgCl2, 3.8°C; (white square) 2 mM MgCl2, 20.8°C; (grey square) 2 mM MgCl2, 37.4°C. All interpolation functions conform to y = axb, with b held between 2.3 and 2.7 (25). (A) Decay times at measured temperature with interpolation plots for each condition. (B) All decay times corrected to 20°C and replotted to verify the behaviour of the linear controls under different temperature conditions: dashed line, no magnesium; solid line, 2 mM MgCl2. Interpolation for two-bend flexibility studies. (C) Circles represent data obtained in the absence of Mg2+; open squares, data with 2 Mg2+.
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Figure 2: Dependence of the linear length (bp) versus τ-value. Interpolations used for single-bend temperature data presented in panels A and B: (black circle) 0 mM MgCl2, 3.8°C; (white circle) 0 mM MgCl2, 20.8°C; (grey circle) 0 mM MgCl2, 37.4°C; (black square) 2 mM MgCl2, 3.8°C; (white square) 2 mM MgCl2, 20.8°C; (grey square) 2 mM MgCl2, 37.4°C. All interpolation functions conform to y = axb, with b held between 2.3 and 2.7 (25). (A) Decay times at measured temperature with interpolation plots for each condition. (B) All decay times corrected to 20°C and replotted to verify the behaviour of the linear controls under different temperature conditions: dashed line, no magnesium; solid line, 2 mM MgCl2. Interpolation for two-bend flexibility studies. (C) Circles represent data obtained in the absence of Mg2+; open squares, data with 2 Mg2+.

Mentions: The method measures the rotational decay times for a linear or heteroduplex DNA or RNA molecule by monitoring the loss of solution birefringence (optical anisotropy) following removal of an orienting electric field pulse. The method exploits the fact that rotational reorientation is extremely sensitive to changes in length of extended molecules in solution (9–11). In a typical experiment, a transient (∼1 μs) electric pulse is applied to the sample cell, which aligns the nucleic acid construct. Once the electric pulse is removed, the solution becomes isotropic as the previously oriented molecules randomize through Brownian motion. For heteroduplex species, the sensitivity of the TEB method can be dramatically increased by extending the helices that flank the central nonhelical structure (Figure 2 of ref. 9). The rate of birefringence decay for a given heteroduplex construct is compared to the decay of a fully duplex species of equivalent axial length. The ratio of the decay times is the outcome of the birefringence experiment, and is related to the apparent angle of the central bend. If the bend in the experimental construct possesses additional flexibility (i.e. more flexibility than an equivalent length of pure helix), this will also lead to a reduction in decay time. To remove this ambiguity, a second nonhelical element is added to the construct; in the current instance, A5 bulge whose angle has been reported elsewhere (12).Figure 2.


Core flexibility of a truncated metazoan mitochondrial tRNA.

Frazer-Abel AA, Hagerman PJ - Nucleic Acids Res. (2008)

Dependence of the linear length (bp) versus τ-value. Interpolations used for single-bend temperature data presented in panels A and B: (black circle) 0 mM MgCl2, 3.8°C; (white circle) 0 mM MgCl2, 20.8°C; (grey circle) 0 mM MgCl2, 37.4°C; (black square) 2 mM MgCl2, 3.8°C; (white square) 2 mM MgCl2, 20.8°C; (grey square) 2 mM MgCl2, 37.4°C. All interpolation functions conform to y = axb, with b held between 2.3 and 2.7 (25). (A) Decay times at measured temperature with interpolation plots for each condition. (B) All decay times corrected to 20°C and replotted to verify the behaviour of the linear controls under different temperature conditions: dashed line, no magnesium; solid line, 2 mM MgCl2. Interpolation for two-bend flexibility studies. (C) Circles represent data obtained in the absence of Mg2+; open squares, data with 2 Mg2+.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553581&req=5

Figure 2: Dependence of the linear length (bp) versus τ-value. Interpolations used for single-bend temperature data presented in panels A and B: (black circle) 0 mM MgCl2, 3.8°C; (white circle) 0 mM MgCl2, 20.8°C; (grey circle) 0 mM MgCl2, 37.4°C; (black square) 2 mM MgCl2, 3.8°C; (white square) 2 mM MgCl2, 20.8°C; (grey square) 2 mM MgCl2, 37.4°C. All interpolation functions conform to y = axb, with b held between 2.3 and 2.7 (25). (A) Decay times at measured temperature with interpolation plots for each condition. (B) All decay times corrected to 20°C and replotted to verify the behaviour of the linear controls under different temperature conditions: dashed line, no magnesium; solid line, 2 mM MgCl2. Interpolation for two-bend flexibility studies. (C) Circles represent data obtained in the absence of Mg2+; open squares, data with 2 Mg2+.
Mentions: The method measures the rotational decay times for a linear or heteroduplex DNA or RNA molecule by monitoring the loss of solution birefringence (optical anisotropy) following removal of an orienting electric field pulse. The method exploits the fact that rotational reorientation is extremely sensitive to changes in length of extended molecules in solution (9–11). In a typical experiment, a transient (∼1 μs) electric pulse is applied to the sample cell, which aligns the nucleic acid construct. Once the electric pulse is removed, the solution becomes isotropic as the previously oriented molecules randomize through Brownian motion. For heteroduplex species, the sensitivity of the TEB method can be dramatically increased by extending the helices that flank the central nonhelical structure (Figure 2 of ref. 9). The rate of birefringence decay for a given heteroduplex construct is compared to the decay of a fully duplex species of equivalent axial length. The ratio of the decay times is the outcome of the birefringence experiment, and is related to the apparent angle of the central bend. If the bend in the experimental construct possesses additional flexibility (i.e. more flexibility than an equivalent length of pure helix), this will also lead to a reduction in decay time. To remove this ambiguity, a second nonhelical element is added to the construct; in the current instance, A5 bulge whose angle has been reported elsewhere (12).Figure 2.

Bottom Line: Thus, the absence of canonical TpsiC-D interactions likely results in greater dispersion of anticodon-acceptor interstem angle than for canonical tRNAs.To test this hypothesis, we have assessed the dispersion of the anticodon-acceptor angle for bovine mtRNA(Ser)(AGY), which lacks the canonical D arm and is thus incapable of forming stabilizing interarm interactions.These results suggest that increased flexibility, in addition to a more open interstem angle, would allow both noncanonical and canonical mtRNAs to utilize the same protein synthetic apparatus.

View Article: PubMed Central - PubMed

Affiliation: National Jewish Health, Denver, CO 80206, USA.

ABSTRACT
Secondary and tertiary structures of tRNAs are remarkably preserved from bacteria to humans, the notable exception being the mitochondrial (m) tRNAs of metazoans, which often deviate substantially from the canonical cloverleaf (secondary) or 'L'-shaped (tertiary) structure. Many metazoan mtRNAs lack either the TpsiC (T) or dihydrouridine (D) loops of the canonical cloverleaf, which are known to confer structural rigidity to the folded structure. Thus, the absence of canonical TpsiC-D interactions likely results in greater dispersion of anticodon-acceptor interstem angle than for canonical tRNAs. To test this hypothesis, we have assessed the dispersion of the anticodon-acceptor angle for bovine mtRNA(Ser)(AGY), which lacks the canonical D arm and is thus incapable of forming stabilizing interarm interactions. Using the method of transient electric birefringence (TEB), and by changing the helical torsion angle between a core mtRNA bend and a second bend of known angle/rigidity, we have demonstrated that the core of mtRNA(Ser)(AGY) has substantially greater flexibility than its well-characterized canonical counterpart, yeast cytoplasmic tRNA(Phe). These results suggest that increased flexibility, in addition to a more open interstem angle, would allow both noncanonical and canonical mtRNAs to utilize the same protein synthetic apparatus.

Show MeSH
Related in: MedlinePlus