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Search for differences in post-transcriptional modification patterns of mitochondrial DNA-encoded wild-type and mutant human tRNALys and tRNALeu(UUR)

Helm, Mark and Florentz, Catherine and Chomyn, Anne and Attardi, Giuseppe (1999) Search for differences in post-transcriptional modification patterns of mitochondrial DNA-encoded wild-type and mutant human tRNALys and tRNALeu(UUR). Nucleic Acids Research, 27 (3). pp. 756-763. ISSN 0305-1048. http://resolver.caltech.edu/CaltechAUTHORS:HELnar99

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Abstract

Post-transcriptional modifications are characteristic features of tRNAs and have been shown in a number of cases to influence both their structural and functional properties, including structure stabilization, amino-acylation and codon recognition. We have developed an approach which allows the investigation of the post-transcriptional modification patterns of human mitochondrial wild-type and mutant tRNAs at both the qualitative and the quantitative levels. Specific tRNA species are long-term labeled in vivo with [P-32]orthophosphate, isolated in a highly selective way, enzymatically digested to mononucleotides and then subjected to two-dimensional thin layer chromatographic analysis. The wild-type tRNA(LyS) and the corresponding tRNALyS carrying the A8344G mutation associated with the MERRF (Myoclonic Epilepsy with Ragged Red Fibers) syndrome exhibit the same modified nucleotides at the same molar concentrations. By contrast, a quantitatively different modification pattern was observed between the wild-type tRNA(LeU(UUR)) and its counterpart carrying the A3243G mutation associated with the MELAS (Mitochondrial Myopathy, Encephalopathy with Lactic Acidosis and Stroke-like episodes) syndrome, the latter exhibiting a 50% decrease in m(2)G content. Complementary sequencing of tRNA(Leu(UUR)) has allowed the localization of this modification at position 10 within the D-stem of the tRNA. The decreased lever of this modification may have important implications for understanding the molecular mechanism underlying the MELAS-associated mitochondrial dysfunction.


Item Type:Article
Additional Information:Copyright © 1999 by Oxford University Press. Received October 12, 1998; Revised and Accepted November 26, 1998. We are grateful to A. Drew and B. Keeley for their excellent technical assistance, J. A. Enriquez and Y. Michikawa for help in preliminary experiments and sequencing of mtDNA, H. Grosjean and H. Brulé for helpful comments and to R. Giegé for constant support and critical reading of the manuscript. This work was supported by NIH grant GM 11726 to G.A., by the Association Française contre la Myopathie (AFM), a NATO Grant for senior scientists to C.F. on sabbatical at Caltech and a Marie Curie fellowship from the EC to M.H.
Subject Keywords:ISOLEUCINE TRANSFER-RNA; ESCHERICHIA-COLI; PROTEIN-SYNTHESIS; HUMAN-CELLS; T-ARM; RESPIRATION; SEQUENCE; MUTATION; MTDNA; AMINOACYLATION
Record Number:CaltechAUTHORS:HELnar99
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:HELnar99
Alternative URL:http://dx.doi.org/10.1093/nar/27.3.756
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:1626
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:07 Feb 2006
Last Modified:26 Dec 2012 08:45

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