A Counterintuitive Mg2+-dependent and Modification-assisted Functional Folding of Mitochondrial tRNAs

Christopher I. Jones, Angela C. Spencer, Jennifer L. Hsu, Linda L. Spremulli, Susan A. Martinis, Michele DeRider, Paul F. Agris

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Mitochondrial tRNAs (mtRNAs) often lack domains and posttranscriptional modifications that are found in cytoplasmic tRNAs. These structural and chemical elements normally stabilize the folding of cytoplasmic tRNAs into canonical structures that are competent for aminoacylation and translation. For example, the dihydrouridine (D) stem and loop domain is involved in the tertiary structure of cytoplasmic tRNAs through hydrogen bonds and a Mg2+ bridge to the ribothymidine (T) stem and loop domain. These interactions are often absent in mtRNA because the D-domain is truncated or missing. Using gel mobility shift analyses, UV, circular dichroism and NMR spectroscopies and aminoacylation assays, we have investigated the functional folding interactions of chemically synthesized and site-specifically modified mitochondrial and cytoplasmic tRNAs. We found that Mg2+ is critical for folding of the truncated D-domain of bovine mtRNAMet with the tRNA's T-domain. Contrary to the expectation that Mg2+ stabilizes RNA folding, the mtRNAMet D-domain structure was unfolded and relaxed, rather than stabilized in the presence of Mg2+. Because the D-domain is transcribed prior to the T-domain, we conclude that Mg2+ prevents misfolding of the 5′-half of bovine mtRNAMet facilitating its correct interaction with the T-domain. The interaction of the mtRNAMet D-domain with the T-domain was enhanced by a pseudouridine located in either the D or T-domains compared to that of the unmodified RNAs (Kd = 25.3, 24.6 and 44.4 μM, respectively). Mg2+ also affected the folding interaction of a yeast mtRNALeu1, but had minimal effect on the folding of an Escherichia coli cytoplasmic tRNALeu. The D-domain modification, dihydrouridine, facilitated mtRNALeu folding. These data indicate that conserved modifications assist and stabilize the formation of the functional mtRNA tertiary structure.

Original languageEnglish (US)
Pages (from-to)771-786
Number of pages16
JournalJournal of Molecular Biology
Volume362
Issue number4
DOIs
StatePublished - Sep 29 2006

Fingerprint

Transfer RNA
Aminoacylation
Pseudouridine
RNA, Transfer, Leu
RNA Folding
Cytoplasmic Structures
Electrophoretic Mobility Shift Assay
Circular Dichroism
Hydrogen
Magnetic Resonance Spectroscopy
Yeasts
RNA
Escherichia coli

Keywords

  • aminoacylation
  • mitochondrial RNA folding
  • tRNA
  • tRNA

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

Cite this

A Counterintuitive Mg2+-dependent and Modification-assisted Functional Folding of Mitochondrial tRNAs. / Jones, Christopher I.; Spencer, Angela C.; Hsu, Jennifer L.; Spremulli, Linda L.; Martinis, Susan A.; DeRider, Michele; Agris, Paul F.

In: Journal of Molecular Biology, Vol. 362, No. 4, 29.09.2006, p. 771-786.

Research output: Contribution to journalArticle

Jones, Christopher I. ; Spencer, Angela C. ; Hsu, Jennifer L. ; Spremulli, Linda L. ; Martinis, Susan A. ; DeRider, Michele ; Agris, Paul F. / A Counterintuitive Mg2+-dependent and Modification-assisted Functional Folding of Mitochondrial tRNAs. In: Journal of Molecular Biology. 2006 ; Vol. 362, No. 4. pp. 771-786.
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AB - Mitochondrial tRNAs (mtRNAs) often lack domains and posttranscriptional modifications that are found in cytoplasmic tRNAs. These structural and chemical elements normally stabilize the folding of cytoplasmic tRNAs into canonical structures that are competent for aminoacylation and translation. For example, the dihydrouridine (D) stem and loop domain is involved in the tertiary structure of cytoplasmic tRNAs through hydrogen bonds and a Mg2+ bridge to the ribothymidine (T) stem and loop domain. These interactions are often absent in mtRNA because the D-domain is truncated or missing. Using gel mobility shift analyses, UV, circular dichroism and NMR spectroscopies and aminoacylation assays, we have investigated the functional folding interactions of chemically synthesized and site-specifically modified mitochondrial and cytoplasmic tRNAs. We found that Mg2+ is critical for folding of the truncated D-domain of bovine mtRNAMet with the tRNA's T-domain. Contrary to the expectation that Mg2+ stabilizes RNA folding, the mtRNAMet D-domain structure was unfolded and relaxed, rather than stabilized in the presence of Mg2+. Because the D-domain is transcribed prior to the T-domain, we conclude that Mg2+ prevents misfolding of the 5′-half of bovine mtRNAMet facilitating its correct interaction with the T-domain. The interaction of the mtRNAMet D-domain with the T-domain was enhanced by a pseudouridine located in either the D or T-domains compared to that of the unmodified RNAs (Kd = 25.3, 24.6 and 44.4 μM, respectively). Mg2+ also affected the folding interaction of a yeast mtRNALeu1, but had minimal effect on the folding of an Escherichia coli cytoplasmic tRNALeu. The D-domain modification, dihydrouridine, facilitated mtRNALeu folding. These data indicate that conserved modifications assist and stabilize the formation of the functional mtRNA tertiary structure.

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KW - tRNA

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