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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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Verification of the expected of secondary structure of the n = 0 construct using chemical modification. (A) The sites of modification are indicated by closed arrows on the extended secondary structure representation. (B) The closed triangles point to sites of modification indicating a single stranded area. The open triangles indicate a site of lesser modification.
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Figure 4: Verification of the expected of secondary structure of the n = 0 construct using chemical modification. (A) The sites of modification are indicated by closed arrows on the extended secondary structure representation. (B) The closed triangles point to sites of modification indicating a single stranded area. The open triangles indicate a site of lesser modification.

Mentions: The secondary structure of the tRNA core and the presence of the A5 bulge were verified by chemical modification probing. DMS alkylates the free N1 of adenosine (A) and, to a lesser extent, the N3 of cytosine (C), when those residues are not involved in Watson–Crick base pairing. Figure 4 shows a probing experiment on the phasing construct, n = 0, where the mtRNA core and A5 bulge are in the closest proximity; alkylation of the A and C bases stops reverse transcriptase one base before the modification, as can be seen in the figure. DMS also alkylates N7 of guanosine, but that modification does not stop reverse transcriptase. Similar experiments on the n = 8 and n = 10 constructs gave similar results, verifying the presence of the A5 bulge and of the stems and loop expected for the tRNA core.Figure 4.


Core flexibility of a truncated metazoan mitochondrial tRNA.

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

Verification of the expected of secondary structure of the n = 0 construct using chemical modification. (A) The sites of modification are indicated by closed arrows on the extended secondary structure representation. (B) The closed triangles point to sites of modification indicating a single stranded area. The open triangles indicate a site of lesser modification.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Verification of the expected of secondary structure of the n = 0 construct using chemical modification. (A) The sites of modification are indicated by closed arrows on the extended secondary structure representation. (B) The closed triangles point to sites of modification indicating a single stranded area. The open triangles indicate a site of lesser modification.
Mentions: The secondary structure of the tRNA core and the presence of the A5 bulge were verified by chemical modification probing. DMS alkylates the free N1 of adenosine (A) and, to a lesser extent, the N3 of cytosine (C), when those residues are not involved in Watson–Crick base pairing. Figure 4 shows a probing experiment on the phasing construct, n = 0, where the mtRNA core and A5 bulge are in the closest proximity; alkylation of the A and C bases stops reverse transcriptase one base before the modification, as can be seen in the figure. DMS also alkylates N7 of guanosine, but that modification does not stop reverse transcriptase. Similar experiments on the n = 8 and n = 10 constructs gave similar results, verifying the presence of the A5 bulge and of the stems and loop expected for the tRNA core.Figure 4.

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